Galanin receptor protein, production and use thereof

ABSTRACT

Galanin receptor proteins, production and use thereof including screening of galanin receptor agonists and antagonists are provided. Galanin receptor proteins, etc. or salts thereof, partial peptides thereof, DNAs coding for the above galanin receptor protein, processes for producing the above receptor protein, methods of screening for a galanin receptor agonist and/or antagonist or screening kits therefor, agonist and/or antagonist compounds or salts thereof obtained by the above screening method or the screening kit, pharmaceutical compositions containing the above compound or its salt, and antibodies against the above receptor protein are provided. It is allowable to efficiently screen a galanin receptor agonist or antagonist by using the galanin receptor protein, the partial peptide thereof, the galanin receptor protein-encoding DNA, the receptor protein-containing cell or its membrane fraction. The pharmaceuticals thus screened or characterized permits various applications including prophylactic and/or therapeutic treatments against a variety of diseases, e.g., stomach ulcer, diabetes, Alzheimer&#39;s disease, dementia, etc. and a sedative.

FIELD OF THE INVENTION

[0001] The present invention relates to novel galanin receptor proteinsand partial peptides thereof; novel DNAs containing a galanin receptorprotein or partial peptide-encoding DNA; processes for producing saidgalanin receptor protein (or partial peptide); use of said receptorprotein (or partial peptide) and said protein (or partialpeptide)-encoding DNA; a method of measuring the physiological actionsof galanin using a galanin receptor protein-expressing cell or thegalanin receptor protein; a method of screening galanin receptoragonists/antagonists using the galanin receptor protein-expressing cellor galanin receptor protein; a kit for said screening; an agonist orantagonist obtained by said screening method; and a pharmaceuticalcomposition containing said agonist or antagonist.

[0002] The present invention also relates to a novel mouse pancreatic 3cell line MIN6-derived galanin receptor protein and a partial peptidethereof; a novel DNA coding for said mouse galanin receptor protein orits partial peptide; processes for producing said mouse galanin receptorprotein or its partial peptide; use of said mouse galanin receptorprotein and said protein or peptide-encoding DNA; a method of measuringthe physiological actions of galanin using a mouse-derived cell lineMIN6 or the mouse galanin receptor protein; and a method of screening agalanin receptor agonist/antagonist using said mouse-derived cell lineMIN6 or the receptor protein.

[0003] The present invention also relates to a novel human galaninreceptor protein; a partial peptide of the human galanin receptorprotein; a novel DNA which codes for the galanin receptor protein orpartial peptide; a vector carrying said DNA; a transformant harboringsaid vector; a process for producing the human galanin receptor protein(or its partial peptide); a method of screening a galanin receptoragonist/antagonist using the human galanin receptor protein or a humangalanin receptor protein-expressing cell (including the transformant); akit for said screening; an agonist or an antagonist, obtained by saidscreening method; and a pharmaceutical composition containing saidagonist or antagonist.

BACKGROUND OF THE INVENTION

[0004] A variety of hormones, neurotransmitters and the like control,regulate or adjust the functions of living bodies via specific receptorslocated in cell membranes. Many of these receptors mediate thetransmission of intracellular signals via activation of a guaninenucleotide-binding protein (hereinafter, sometimes referred to as “Gprotein”) with which the receptor is coupled and possess the common(homologous) structure, i.e. seven transmembranes (membrane-spanningregions (domains)). Therefore, such a receptor is generically referredto as “G protein coupled receptor” or “seven transmembrane(membrane-spanning) receptor”.

[0005] G protein coupled receptor proteins which are widely distributedin the functional cellular surface of cells and organs in the livingbodies have a very important role as targets for molecules such ashormones, neurotransmitters and physiologically active substances, whichmolecules control, regulate or adjust the functions of living bodies.

[0006] The pancreas plays an important role of carrying out thecarbohydrate metabolism by secreting not only a digestive fluid but alsoglucagon and insulin. Insulin is secreted from the β cells and itssecretion is promoted chiefly by glucose. It has been known that avariety of receptors exist in the β cells, and the secretion of insulinis controlled by various factors such as peptide hormones (galanin,somatostatin, gastric inhibitory polypeptide, glucagon, amylin, etc.),sugars (mannose, etc.), amino acids, and neurotransmitters in additionto glucose. As for the galanin and amylin, however, there has not yetbeen reported any discovery concerning the structure of their receptorprotein cDNA. It is not known whether there exist any unknown receptorproteins or receptor protein subtypes.

[0007] It is a very important means in investigating development of newpharmaceuticals to clarify the relation between substances controllingthe complicated functions of pancreas and specific receptors thereto. Inorder to develop new pharmaceuticals by conducting an effectivescreening of agonists and antagonists to the receptor proteins forcontrolling the functions of pancreas, it was necessary to investigatethe function of receptor protein genes and also to express them in asuitable expression system.

[0008] By utilizing the fact that a G protein coupled receptor proteinexhibits homology in part of the structure thereof at the amino acidsequence level, an experiment of looking at DNAs coding for novelreceptor proteins relying upon a polymerase chain reaction (hereinaftersimply referred to as “PCR”) has recently been made.

[0009] Galanin is a peptide existing in central and peripheral areasand, in central area, it shows an action of inhibition of liberation ofneurotransmitter (acetylcholine) (European Journal of Pharmacology,vol.164, 355-360, 1989) and an action of antagonizing foreignacetylcholine (Proceedings of National Academy of Sciences, U.S.A.,vol.85, 9841-9845, 1988) while, in pancreas, it shows a pharmacologicalaction such as inhibition of insulin secretion (Diabates, vol. 34,192-196, 1985). It has been also confirmed that galanin has an effect ofinhibiting the behavior of learning (Neuroscience Letters, vol.88,331-335, 1988) and of inhibiting the feeling of fullness after a meal.Such findings suggest a possibility that, if pharmaceuticals whichinhibit the action of galanin are developed, they may be used asintelligence tropic agents and as remedies for obesity and for diabetes.

[0010] All of the pharmacological actions of galanin take place via aspecific galanin receptor existing in target tissues. Accordingly, thesimplest means for inhibiting the action of galanin is to developpharmaceuticals which specifically inhibit the reaction of galanin withthe receptor, i.e. galanin receptor antagonists. In the development ofgalanin receptor antagonists, it is usually necessary to conduct areceptor binding experiment. In the case of galanin, experiments ongalanin receptor binding using membrane fractions of brain hippocampalformation (European Journal of Biochemistry, vol. 181, 269-276, 1989)and of stomach and duodenum (Peptides, vol. 11, 333-338, 1990) have beenreported already.

[0011] It has been also reported that there is a specific galaninreceptor in Rin-m-5F cells obtained from rat pancreas (Endocrinology,vol. 124, 2635-2641, 1989). According to the above-mentioned reports, itis already possible to conduct a galanin receptor binding experiment.However, the amount of the galanin receptor in those membrane fractionsis as low as around 50 fmol/mg and, therefore, it was necessary to use alarge amount of cell fractions for one measurement.

[0012] Galanin exhibits the above-mentioned pharmacological actions inliving body and, if the actions can be easily measured in vitro, thatwill be meaningful for the process of developing the receptorantagonists. It has been reported already that the action of inhibitingthe insulin secretion by galanin can be substituted with an in vitromeasurement using Langerhans islet isolated from pancreas (EuropeanJournal of Pharmacology, Vol. 203, 111-114, 1991). However, Langerhansisland is required to be isolated upon each experiment and, therefore,this method is not easily accomplished.

[0013] As easier means, several methods using pancreatic β-cell strains(Rin-m-5F cells) have reported. They are, for example, a method in whichan effect of galanin receptor to a second messenger system (i.e. anactivity of inhibiting the adenylate cyclase) is measured (EuropeanJournal of Biochemistry, Vol. 177, 147-152, 1988) and a method in whichan activity of opening the potassium channel is measured (Proceedings ofNational Academy of Sciences, U.S.A., Vol. 85, 1312-1316, 1988). Amethod in which the activity of inhibiting the insulin secretion ofgalanin using said cell strain has been reported too. However, thosemethods are applicable only for insulin secretion which is dependentupon forskolin (an adenylate cyclase activator) and the measurement forsecretion of glucose-dependent insulin is not possible. Further, thesecretion amount of insulin is small and the sensitivity is low.

[0014] After those, a method of preparing the β-cell strains usingpancreas of transgenic mice (Proceedings of National Academy ofSciences, U.S.A., Vol. 85, 9037-9041, 1988) has been developed andestablishments of cell strains such as β TC-1 cells (Proceedings ofNational Academy of Sciences, U.S. A., Vol. 85, 9037-9041, 1988), IgSV195 cells (Diabates, Vol. 38, 1056-1062, 1989) and MIN6 cells(Endocrinology, Vol. 127, 126-132, 1990) have been reported. Amongthose, MIN6 cells hold the ability of insulin secretion depending uponthe glucose concentration (which is a differentiating function inherentto β-cells) in the best manner and, in addition, they secret insulin ina high amount. However, it has not been known yet that galanin receptorprotein is expressed in said MIN6 cells. In addition, there has been noproposal yet for an evaluating system for the biological activity ofgalanin and also for an effective method for screening the galaninreceptor agonist or antagonist using the MIN6 cells.

[0015] Recently, cDNA which codes for human galanin receptor protein wascloned and its nucleotide sequence and also its amino acid sequenceencoded by said cDNA have been disclosed (Proceedings of NationalAcademy of Sciences, U.S.A., Vol. 91, 9780-9783, Oct. 11, 1994).However, there is no disclosure at all for a specific means forscreening the galanin receptor agonist/antagonist using said receptor.Under such circumstances, a method for screening and assessing galaninreceptor agonist/antagonist in an efficient manner is still desired.

[0016] Galanin is a polypeptide comprising 29 amino acid residuesseparated from porcine small intestine [Tatemoto, K. et al., FEBSLetter, 164, 124-128(1983)] and its primary structure is hardly similarto those of other brain and intestinal hormones. Galanin immunoactivityis widely distributed in central nervous system and peripheral nervoussystem together with its receptor [Scofitsch, G. and Jacobowitz, D. M.,Peptides, 6, 509-546(1985); Melander, T. et al., Journal of ComparativeNeurology, 248, 475-517(1986); Rokaeus, A., Trends in Neuroscience,10,158-164(1987)] and, since its distribution pattern is identical withthe region containing the traditional neurotransmitters such as 5-HT,noradrenaline and acetylcholine, it is likely that galanin is presenttogether with such neurotransmitters and controls the prenervous andpostnervous actions by those neurotransmitters.

[0017] Galanin has many physiological actions and, in central nervoussystem, it strongly inhibits the single synaptic reflection in spinalnerve [Yanagisawa, M. et al., Neuroscience Letter, 70, 278-282(1986)]and its action is known to be far stronger than somatostatin. Inaddition, the physiological importance of galanin in nerve center hasbeen greatly suggested because of stimulation of action for taking food[Kyrokouli, S. E. et al., European Journal of Pharmacology, 122,159-160(1986)], participation in formation of memory [Crawley, J. N. andWenk, G. L., Trends in Neuroscience, 12,278-282(1989)], inhibition ofdopamine in median elevation [Nordstrom, O. et al., Neuroscience Letter,73, 21-26(1987)], inhibition of release of acetylcholine in hippocampaldouble sides [Fisone, G. et al., Proceedings of the National Academy ofSciences of U. S. A., 84, 7339-7343(1987)], a decrease in metaboliccirculation of 5-HT [Fuxe K., et al., Acta. Physiol. Scand., 133,579-581(1988)] and a decrease in a glutamic acid release by activationof ATP-sensitive K⁺ channel [Ben-Ari, Y., European Journal ofNeuroscience, 2, 62-68(1990)] as a result of administration of galaninto paraventricular nucleus of rats.

[0018] Especially, galanin is an only neuropeptide in which cholineacetyltransferase is coexisting in the medial septal nucleus, nucleus ofdiagonal band and basal nucleus [Melender, T. et al., Brain Research,360, 130-138(1985); Melender, T. et al., Neuroscience Letter, 19,223-240(1986); Chen-Palay, V., Brain Research Bulletin, 21,465-472(1988)] and is known to act on cholinergic nerves in aninhibiting manner while, on the other hand, it is expected that, sincedenaturation in cholinergic nerves is noted in those sites inAlzheimer's disease, galanin antagonist may prevent the denaturation ofthe cholinergic nerves in Alzheimer's disease or the like [Whitehouse,P. J., et al., Science, 215, 1237-1239(1982); Chen-Palay, V., Journal ofComparative Neurology, 273-543-557 (1988)]. In hypophysis, action ofstimulating the secretion of growth hormones and prolactin has beennoted [Tanoh, T., et al., Neuroendocrinology, 54, 83-88(1991);Koshiyama, H., et al., Neuroscience Letter, 75, 49-54(1987)].Particularly in the secretion of growth hormones, participation ofcholinergic neuron via adjustment of secretion of hypothalamicsomatostatin is noted.

[0019] On the other hand, in peripheral systems, galanin inhibits thebasal secretion of insulin both in vivo and in vitro [McDnald, T. J. etal., Diabates, 34, 192-196(1985); Takeda, Y. et al., BiomedicalResearch, 8 (Suppl.), 117-125 (1987); Lindskog, S. et al., Acta.Physiol. Scand., 129, 305-309(1987)] and, in addition, it inhibits therelease of insulin by stimulation of glucose [Dunning, B. E. andTaborsky, G. J., Jr., Diabates, 37, 1157-1162(1988)]. When furtherimmunohistological observation that nerve fiber net containing a densegalanin immunoactivity is noted around Langerhans islet of β cells istaken into consideration, it has been strongly suggested that galanin isone of the nerve controlling factors for secretion of pancreatichormones, especially insulin. It is also noted that, in stomach, galanininhibits the basal secretion of somatostatin on a dose-depending manneror it inhibits the secretion of somatostatin or gastrin by stimulationof GRP and that nerve fiber net containing galanin immunoactivity isobserved in stomach and, accordingly, it is suggested that, even instomach, galanin acts as one of the important nerve controlling factorsfor adjusting the secretion in stomach [Yanaihara, N. et al., in“Galanin” (ed. by Hokfelt, T. et al.), Macmillan Press, 185-196(1991)].

[0020] From the above descriptions, it is understood that galaninagonist is useful as a pharmaceutical agent such as a stimulant forsecretion of growth hormones and an inhibitor for secretion of insulinand that galanin antagonist is useful as another pharmaceutical agentsuch as an inhibitor for secretion of growth hormones and a stimulantfor secretion of insulin.

[0021] Usually, in developing agonists and antagonists forphysiologically-active substances, investigations are made on thecompounds which have high affinity with the receptors to which saidsubstance is specifically bonded. At present, bovine hippocampalmembrane fraction is used as a galanin receptor but, because of thedifference in the animal species used, there is no guarantee that thecompound exhibiting a high affinity to said membrane fraction has a highaffinity to human galanin receptor as well. Human galanin receptor cDNAhas been cloned and reported to exhibit an expression in COS cells[Habert-Ortoll, E. et al., Proceedings of the National Academy ofSciences, U. S. A., 91, 9780-9783(1994)] but, since the expressed amountis small and the expression is mere transient, it is thought to beunsuitable for screening.

SUMMARY OF THE INVENTION

[0022] One object of the present invention is to provide novel galaninreceptor proteins and partial peptides thereof or salts thereof; DNAscomprising a DNA coding for said galanin receptor protein or its partialpeptide; vectors carrying said DNA; transformants harboring said vector;cell membrane fractions obtained from said transformant; processes forproducing said receptor protein or its partial peptide, or a saltthereof; methods for measuring the physiological actions of galaninusing the galanin receptor protein (including a cell membrane fractioncontaining the receptor protein) or a galanin receptorprotein-expressing cell (including the transformant); screening methodsfor a galanin receptor agonist/antagonist using the galanin receptorprotein or a galanin receptor protein-expressing cell (including thetransformant); kits for said screening; agonists or antagonists,obtained by said screening method; pharmaceutical compositionscontaining said agonist or antagonist; antibodies against said receptorprotein; immunoassays using said receptor protein or said antibody; anduse of said receptor protein and encoding DNA.

[0023] Another object of the present invention is to provide novel mousepancreatic β cell line MIN6-derived galanin receptor proteins or partialpeptides thereof; DNAs comprising a DNA coding for said galanin receptorprotein or partial peptide; processes for producing said receptorprotein or its partial peptide; methods of measuring the physiologicalactions of galanin using a mouse-derived cell line MIN6 or the galaninreceptor protein; screening methods for a galanin receptoragonist/antagonist using said mouse-derived cell line MIN6 or thereceptor protein; antibodies against said receptor protein; immunoassaysusing said receptor protein or said antibody; and use of said galaninreceptor protein or said receptor protein peptide-encoding DNA.

[0024] Human galanin receptor proteins manufactured by the conventionalmethod and the COS cells which express said human galanin receptorprotein are insufficient as receptor samples for conducting a screeningfor galanin receptor agonist/antagonist. Consequently, there has been ademand for developing a more practical method for manufacturing humangalanin receptor proteins.

[0025] If it is possible to screen the agonist/antagonist of galaninreceptor using human galanin receptor protein, it is now possible toovercome the disadvantage by the use of experimental animals (forexample, the possibility that, due to a difference in species, compoundswhich do not achieve an effect to human being may be obtained) wherebyit is expected to conduct a development of pharmaceutical agentseffective to human being in an efficient manner.

[0026] Yet another object of the present invention is to provide novelhuman galanin receptor proteins; partial peptides of the human galaninreceptor protein; novel DNAs which code for the galanin receptor proteinor partial peptide; vectors carrying said DNA; transformants harboringsaid vector; cell membrane fractions obtained from said transformant;processes for producing the human galanin receptor protein (or itspartial peptide); methods for measuring the physiological actions ofgalanin using a human galanin receptor protein-expressing cell, saidhuman galanin receptor protein; screening methods for a galanin receptoragonist/antagonist using a human galanin receptor protein-expressingcell (including the transformant); kits for said screening; agonists orantagonists, obtained by said screening method; pharmaceuticalcompositions containing said agonist or antagonist; antibodies againstsaid human galanin receptor protein; immunoassays using said receptorprotein or said antibody; and use of said human galanin receptor proteinand encoding DNA.

[0027] In order to achieve the above-mentioned aims, the presentinventors have made extensive investigations. As a result, the presentinventors have succeeded in synthesizing DNA primers effective inefficiently isolating DNAs (DNA fragments) coding for G protein coupledreceptor proteins by PCR techniques. The present inventors havesucceeded in amplifying cDNA derived from various cells with saidsynthetic DNA primer, and have forwarded the analysis. Thus, the presentinventors have succeeded in isolating novel G protein coupled receptorprotein-encoding cDNAs, in determining the partial structure thereof,and have considered that the isolated cDNAs are homologous to known Gprotein coupled receptors at the nucleotide sequence level and at theamino acid sequence level and are each coding for a novel galaninreceptor protein. Based upon the above knowledge, the present inventorshave discovered that these DNAs make it possible to obtain a cDNA havinga full length open reading frame (ORF) of the receptor protein, hence,to produce the receptor protein. The inventors have further succeeded insequencing an entire amino acid sequence and entire nucleotide sequenceof said galanin receptor protein.

[0028] The present inventors have found that, when said receptor proteinexpressed by a suitable means is used, an agonist or an antagonist tosaid receptor protein can be screened in vivo or from natural ornonnatural compounds by a receptor protein binding experiment or by ameasurement of intracellular second messenger as an index. The presentinventors have further found that said agonist and antagonist can bedeveloped as preventive and therapeutic agents for the diseases orsymptoms related to or caused by galanin.

[0029] The present inventors have furthermore found that the glucose orforskolin-dependent insulin secretion in cells expressing said galaninreceptor protein is inhibited by galanin. That has been a finding forthe first time. Depending upon said finding, the present inventors havefound an easy and simple method for measuring the activity of galaninand galanin antagonist. At the same time, the present inventors havealso found that the cell membrane fractions of cells expressing saidgalanin receptor protein contain large amount of galanin receptors andsucceeded in establishing a screening for galanin receptoragonist/antagonist using the cell membrane fractions thereof.

[0030] For example, the present inventors have amplified G proteincoupled receptor protein-encoding cDNA derived from mouse pancreaticβ-cell strain MIN6 using a synthetic DNA primer for more effectiveisolation thereof, whereby its analysis has been carried out.

[0031] As a result thereof, the present inventors have succeeded inisolating the mouse-derived cDNA fragment which codes for a novel Gprotein coupled receptor protein and in elucidating its partialstructure. In said mouse-derived G protein coupled receptor protein,there are similarities (homologies) at DNA and amino acid levels to theknown G protein coupled receptor and, therefore, it is believed that itcodes for a novel receptor protein exhibiting an expressing function inmouse pancreas.

[0032] The present inventors further continued their studies and havesucceeded in cloning cDNA having a full-length translation unit and inanalyzing an entire amino acid sequence and an entire nucleotidesequence of said receptor protein. Since said mouse-derived G proteincoupled receptor protein has a high homology at DNA and amino acidlevels to the human-derived galanin receptor protein (Proceedings ofNational Academy of Sciences, U.S.A., 91, 9780-9783, 1994), it has beenfound that said mouse-derived G protein coupled receptor protein isidentical with a mouse-derived galanin receptor protein.

[0033] Furthermore, the present inventors have newly found that theglucose or forskolin-dependent insulin secretion of MIN6 cells isinhibited by galanin. Based upon said finding, the present inventorshave found an easy and simple method for measuring the activity ofgalanin and galanin antagonist. At the same time, the present inventorshave also found that the cell membrane fractions of MIN6 cells containlarge amount (0.5-1.0 pmol/mg) of galanin receptor and succeeded inestablishing a method of screening galanin receptor agonist/antagonistusing the cell membrane fractions of MIN6 cells.

[0034] To be more specific, the present inventors have amplified andcloned novel cDNA fragments derived from mouse pancreatic β cell strainMIN6 as shown in FIG. 1 by PCR and, from the result of analysis of theirsequence, have clarified that they code for a novel receptor protein.When said sequence was translated into amino acid sequences, third,fourth, fifth and sixth transmembrane domains were confirmed onhyrophobic plots (FIG. 2). The size of the amplified DNA was about 400bp which was almost same as that of the known G protein coupled receptorprotein.

[0035] The inventors have retrieved the data base based on, as atemplate, the nucleotide sequence of the isolated DNA and observed 36%homology to human-derived somatostatin receptor subtype 4 (JN0605), 30%homology to human-derived somatostatin receptor subtype 2 (B41795), and30% homology to rat-derived ligand unknown receptor (A39297),respectively (FIG. 3), which are known G protein coupled receptorproteins. The aforementioned abbreviations in parentheses are referencenumbers that are assigned when they are registered as data toNBRF-PIR/Swiss-PROT and are, usually, each called “Accession Number” or“Entry Name”.

[0036] Moreover, the present inventors have prepared cDNA from thepoly(A)⁺ RNA fractions extracted from MIN6 cells and have inserted saidcDNA into lambda gt22 phage to prepare a cDNA library. Further, thepresent inventors have screened the cDNA library using, as a probe, theG protein coupled receptor protein cDNA fragment p3H2-34 obtained by PCRand succeeded in cloning cDNA which completely codes for the G proteincoupled receptor protein of the present invention. A nucleotide sequenceof said cDNA and an amino acid sequence encoded thereby are given inFIG. 4. A hydrophobic plotting was conducted based upon said amino acidsequence and the first, second, third, fourth, fifth, sixth and seventhtransmembrane domains were confirmed (FIG. 5). The G protein coupledreceptor protein of the present invention has 92% homology at the aminoacid level to the known human galanin receptor protein.

[0037] In another aspect, the present inventors have succeeded, forexample, in cloning a DNA which codes for novel human galanin receptorprotein having an amino acid sequence which differs from that of knownhuman galanin receptor protein. In the known human galanin receptorprotein, the fifteenth amino acid in its amino acid sequence is Cyswhile, in the human galanin receptor protein of the present invention,the fifteenth amino acid in its amino acid sequence (SEQ ID NO: 5 andFIGS. 12 & 13) is Trp. In addition, in the nucleotide sequence of DNAwhich codes for the known human galanin receptor protein, the basesequence which codes for the fifteenth amino acid of said human galaninreceptor protein is ¹⁵Cys (TGT) while, in the base sequence of DNA whichcodes for the human galanin receptor protein of the present invention,the base sequence which codes for the fifteenth amino acid in said humangalanin receptor protein is ¹⁵Trp (TGG).

[0038] The present inventors have further succeeded in manufacturing aCHO cell strain which expresses far more amount of the human galaninreceptor protein of the present invention than the COS cells whichexpress the known human galanin receptor protein [Habert-Ortoll, E. etal., Proceedings of the National Academy of Sciences of the U. S. A.,91, 9780-9783 (1994)]. It has been furthermore found that, when said CHOcell strain of the human galanin receptor protein of the presentinvention or partial peptide thereof is used, it is now possible toscreen the human galanin receptor agonist/antagonist in an effective andreliable manner. Based upon those findings, the present inventors havecontinued various investigations and, as a result, they have achievedthe present invention.

[0039] Accordingly, one aspect of the present invention is

[0040] (1) a galanin receptor protein comprising an amino acid sequenceselected from the group consisting of an amino acid sequence representedby SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5 and substantialequivalents thereto, or a salt thereof;

[0041] (2) the receptor protein according to the above (1), which isproduced by a transformant CHO cell;

[0042] (3) a DNA which comprises a nucleotide sequence coding for agalanin receptor protein of the above (1);

[0043] (4) a vector comprising the DNA according to the above (3);

[0044] (5) a transformant carrying the vector according to the above(4);

[0045] (6) the transformant according to the above (5), wherein the hostcell is a CHO cell;

[0046] (7) a process for producing a galanin receptor protein accordingto the above (1), which comprises culturing a transformant of the above(5) under conditions suitable to express said galanin receptor protein;

[0047] (8) a screening method for an agonist or antagonist of a galaninreceptor protein according to the above (1), which comprises carryingout a comparison between:

[0048] (i) at least one case where galanin is contacted with at leastone component selected from the group consisting of a galanin receptorprotein according to the above (1), a partial peptide thereof and amixture thereof, and

[0049] (ii) at least one case where galanin together with a compound tobe tested is contacted with at least one component selected from thegroup consisting of a galanin receptor protein according to the above(1), a partial peptide thereof and a mixture thereof;

[0050] (9) a kit for the screening of one or more agonists orantagonists to a galanin receptor protein according to the above (1),which comprises at least one component selected from the groupconsisting of a galanin receptor protein according to the above (1), apartial peptide thereof and a mixture thereof; and

[0051] (10) an agonist or antagonist of a galanin receptor, which isobtained by the screening method according to the above (8) or by thekit according to the above (9).

[0052] Another aspect of the present invention is

[0053] (11) a mouse-derived galanin receptor protein comprising an aminoacid sequence selected from the group consisting of an amino acidsequence represented by SEQ ID NO: 1 and substantial equivalentsthereto; or a salt thereof;

[0054] (12) a mouse-derived galanin receptor protein according to theabove (11), which comprises an amino acid sequence selected from thegroup consisting of an amino acid sequence represented by SEQ ID NO: 2and substantial equivalents thereto; or a salt thereof;

[0055] (13) a human galanin receptor protein comprising an amino acidsequence selected from the group consisting of an amino acid sequencerepresented by SEQ ID NO: 5 and substantial equivalents thereto; or asalt thereof;

[0056] (14) a partial peptide of a galanin receptor protein according tothe above (1), or a salt thereof;

[0057] (15) a partial peptide of a mouse-derived galanin receptorprotein according to the above (11) or (12), or a salt thereof;

[0058] (16) a partial peptide of a human galanin receptor proteinaccording to the above (13), or a salt thereof;

[0059] (17) a DNA which comprises a nucleotide sequence coding for amouse-derived galanin receptor protein of the above (11) or (12);

[0060] (18) a DNA which comprises a nucleotide sequence coding for ahuman galanin receptor protein of the above (13);

[0061] (19) a DNA of the above (17) comprising a nucleotide sequencerepresented by SEQ ID NO: 3; 1

[0062] (20) a DNA of the above (17) comprising a nucleotide sequencerepresented by SEQ ID NO: 4;

[0063] (21) a DNA of the above (18) comprising a nucleotide sequencerepresented by SEQ ID NO: 6;

[0064] (22) a vector comprising a DNA according to the above (17);

[0065] (23) a vector comprising a DNA according to the above (18);

[0066] (24) a transformant (including a transfectant) carrying a vectorof the above (22);

[0067] (25) a transformant (including a transfectant) carrying a vectorof the above (23);

[0068] (26) a process for producing a mouse-derived galanin receptorprotein or a salt thereof according to the above (11), which comprisesculturing a transformant of the above (24) to produce said galaninreceptor on the membrane of the transformant;

[0069] (27) a process for producing a human galanin receptor protein ora salt thereof according to the above (13), which comprises culturing atransformant of the above (25) under conditions to express said galaninreceptor;

[0070] (28) a cell or membrane fraction containing a galanin receptorprotein according to the above (1);

[0071] (29) a cell or membrane fraction containing a mouse-derivedgalanin receptor protein according to the above (11) or (12);

[0072] (30) a cell or membrane fraction containing a human galaninreceptor protein according to the above (13);

[0073] (31) a screening method for a galanin receptor agonist and/orantagonist, which comprises using a galanin receptor protein accordingto the above (1), a partial peptide according to the above (14) or acell or membrane fraction according to the above (28);

[0074] (32) a screening method for a mouse-derived galanin receptoragonist and/or antagonist, which comprises using a mouse-derived galaninreceptor protein according to the above (11) or (12), a partial peptideaccording to the above (15) or a cell or membrane fraction according tothe above (29);

[0075] (33) a screening method for a human galanin receptor agonistand/or antagonist, which comprises using a human galanin receptorprotein according to the above (13), a partial peptide according to theabove (16) or a cell or membrane fraction according to the above (30);

[0076] (34) a screening method for a galanin receptor agonist and/orantagonist, which comprises carrying out a comparison between:

[0077] (i) at least one case where galanin is contacted with at leastone component selected from the group consisting of a galanin receptorprotein or a salt thereof according to the above (1), a partial peptideor a salt thereof according to the above (14), a cell or membranefraction according to the above (28), and a mixture thereof, and

[0078] (ii) at least one case where galanin together with a sample(including a compound) to be tested is contacted with at least onecomponent selected from the group consisting of a galanin receptorprotein or a salt thereof according to the above (1), a partial peptideor a salt thereof according to the above (14), a cell or membranefraction according to the above (28), and a mixture thereof;

[0079] (35) a screening method for a mouse-derived galanin receptoragonist and/or antagonist, which comprises carrying out a comparisonbetween:

[0080] (i) at least one case where galanin is contacted with at leastone component selected from the group consisting of a mouse-derivedgalanin receptor protein or a salt thereof according to the above (11),a partial peptide or a salt thereof according to the above (15), and amixture thereof, and

[0081] (ii) at least one case where galanin together with a sample(including a compound) to be tested is contacted with at least onecomponent selected from the group consisting of a mouse-derived galaninreceptor protein or a salt thereof according to the above (11), apartial peptide or a salt thereof according to the above (15), and amixture thereof;

[0082] (36) a screening method for a human galanin receptor agonistand/or antagonist, which comprises carrying out a comparison between:

[0083] (i) at least one case where galanin is contacted with at leastone component selected from the group consisting of a human galaninreceptor protein or a salt thereof according to the above (13), apartial peptide or a salt thereof according to the above (16), a cell ormembrane fraction according to the above (30), and a mixture thereof,and

[0084] (ii) at least one case where galanin together with a sample(including a compound) to be tested is contacted with at least onecomponent selected from the group consisting of a human galanin receptorprotein or a salt thereof according to the above (13), a partial peptideor a salt thereof according to the above (16), a cell or membranefraction according to the above (30), and a mixture thereof;

[0085] (37) a kit for the screening of a galanin receptor agonist and/orantagonist, which comprises at least one component selected from thegroup consisting of a galanin receptor protein or a salt thereofaccording to the above (1), a partial peptide or a salt thereofaccording to the above (14), a cell or membrane fraction according tothe above (28), and a mixture thereof;

[0086] (38) a kit for the screening of a mouse-derived galanin receptoragonist and/or antagonist, which comprises at least one componentselected from the group consisting of a mouse-derived galanin receptorprotein or a salt thereof according to the above (11) or (12), a partialpeptide or a salt thereof according to the above (15), a cell ormembrane fraction according to the above (29), and a mixture thereof;

[0087] (39) a kit for the screening of a human galanin receptor agonistand/or antagonist, which comprises at least one component selected fromthe group consisting of a galanin receptor protein or a salt thereofaccording to the above (13), a partial peptide or a salt thereofaccording to the above (16), a cell or membrane fraction according tothe above (30), and a mixture thereof;

[0088] (40) a galanin receptor agonist and/or antagonist, obtained by amethod according to any of the above (31) to (36) or a kit according toany of the above (37) to (39);

[0089] (41) a galanin receptor agonist and/or antagonist, obtained by amethod according to the above (32) or (35) or a kit according to theabove (38);

[0090] (42) a galanin receptor agonist and/or antagonist, obtained by amethod according to the above (33) or (36) or a kit according to theabove (39);

[0091] (43) a pharmaceutical composition comprising an effective amountof the galanin receptor agonist according to (40);

[0092] (44) a pharmaceutical composition comprising an effective amountof the galanin receptor agonist according to (41);

[0093] (45) a pharmaceutical composition comprising an effective amountof the galanin receptor agonist according to (42);

[0094] (46) a pharmaceutical composition comprising an effective amountof the galanin receptor antagonist according to (40);

[0095] (47) a pharmaceutical composition comprising an effective amountof the galanin receptor antagonist according to (41);

[0096] (48) a pharmaceutical composition comprising an effective amountof the galanin receptor antagonist according to (42);

[0097] (49) a pharmaceutical composition according to (43) which is aninhibitor for acetylcholine liberation, an inhibitor for insulinsecretion, a stimulant for growth hormone secretion, an inhibitor forlearning behavior or satiety;

[0098] (50) a pharmaceutical composition according to (46) which is anagent for promoting the acetylcholine liberation, an agent forinhibiting the growth hormone secretion, an agent for promoting theinsulin secretion, an agent for promoting the learning behavior or anagent for promoting satiety;

[0099] (51) an antibody against at least one component selected from thegroup consisting of a galanin receptor protein or a salt thereofaccording to the above (1) and a partial peptide or a salt thereofaccording to the above (14);

[0100] (52) an antibody against at least one component selected from thegroup consisting of a mouse-derived galanin receptor protein or a saltthereof according to the above (11) or (12) and a partial peptide or asalt thereof according to the above (15); and

[0101] (53) an antibody against at least one component selected from thegroup consisting of a human galanin receptor protein or a salt thereofaccording to the above (13) and a partial peptide or a salt thereofaccording to the above (16).

[0102] To be more specific, the present invention relates to thefollowing:

[0103] (54) a method of screening a galanin receptor agonist orantagonist, characterized in that, the binding amount of the labeledgalanin with the galanin receptor protein or its salt according to (1)(e.g., the mouse-derived galanin receptor protein or its salt accordingto (11), etc.) or with the partial peptide or its salt according to (14)(e.g., the partial peptide of the mouse-derived galanin receptor proteinor its salt according to (15), etc.) is measured in the case where thelabeled galanin is contacted with the galanin receptor protein or itssalt according to (1) (e.g., the mouse-derived galanin receptor proteinor its salt according to (11), etc.) or with the partial peptide or itssalt according to (14) (e.g., the partial peptide of the mouse-derivedgalanin receptor protein or its salt according to (15), etc.) and alsoin the case where the labeled galanin and the test compound arecontacted with the galanin receptor protein or its salt according to (1)(e.g., the mouse-derived galanin receptor protein or its salt accordingto (11), etc.) or with the partial peptide or its salt according to (14)(e.g., the partial peptide of the mouse-derived galanin receptor proteinor its salt according to (15), etc.) and the comparison is made betweenthem;

[0104] (55) a method of screening a galanin receptor agonist orantagonist, characterized in that, the labeled galanin is contacted withthe cells (except mouse-derived MIN6 cells [FERM BP-4954]) containingthe galanin receptor protein according to (1) (e.g., the mouse-derivedgalanin receptor protein according to (11), etc.) and the labeledgalanin and the test compound are contacted with the cells (exceptmouse-derived MIN6 cells [FERM BP-4954]) containing the galanin receptorprotein according to (1) (e.g., the mouse-derived galanin receptorprotein according to (11), etc.) and the binding amounts of the labeledgalanin with said cells in both cases are measured and compared;

[0105] (56) a method of screening a galanin receptor agonist orantagonist, characterized in that, the labeled galanin is contacted withthe cell membrane fractions of cells (except mouse-derived MIN6 cells[FERM BP-4954]) containing the galanin receptor protein according to (1)(e.g., the mouse-derived galanin receptor protein according to (11),etc.) and the labeled galanin and the test compound are contacted withthe cell membrane fraction of cells (except mouse-derived MIN6 cells[FERM BP-4954]) containing the galanin receptor protein according to (1)(e.g., the mouse-derived galanin receptor protein according to (11),etc.) and the binding amounts of the labeled galanin with the membranefractions of said cells in both cases are measured and compared;

[0106] (57) a method of screening a galanin receptor agonist orantagonist, characterized in that, the labeled galanin is contacted withthe galanin receptor protein according to (1) (e.g., the mouse-derivedgalanin receptor protein according to (11), etc.) expressed in cellmembranes of the transformant according to (5) (e.g., the mouse-derivedgalanin receptor protein-expressible transformant according to (24),etc.) by culturing said transformant and the labeled galanin and thetest compound are contacted with the galanin receptor protein accordingto (1) (e.g., the mouse-derived galanin receptor protein according to(11), etc.) expressed in cell membranes of the transformant according to(5) (e.g., the mouse-derived galanin receptor protein-expressibletransformant according to (24), etc.) by culturing said transformant andthe binding amounts of the labeled galanin with said galanin receptor inboth cases are measured and compared;

[0107] (58) a method of screening a galanin receptor agonist orantagonist, characterized in that, galanin is contacted with the cells(except the mouse-derived MIN6 cells [FERM BP-4954]) containing thegalanin receptor protein according to (1) (e.g., the mouse-derivedgalanin receptor protein according to (11), etc.) and galanin and thetest compound are contacted with the cells (except the mouse-derivedMIN6 cells [FERM BP-4954]) containing the galanin receptor proteinaccording to (1) (e.g., the mouse-derived galanin receptor proteinaccording to (11), etc.) and the resulting cell-stimulating activitiesvia the galanin receptor protein in both cases are measured andcompared;

[0108] (59) a method of screening a galanin receptor agonist orantagonist, characterized in that, galanin is contacted with the galaninreceptor protein according to (1) (e.g., the mouse-derived galaninreceptor protein according to (11), etc.) expressed in cell membranes ofthe transformant according to (5) (e.g., the mouse-derived galaninreceptor protein-expressible transformant according to (24), etc.) byculturing said transformant and galanin and the test compound arecontacted with the galanin receptor protein according to (1) (e.g., themouse-derived galanin receptor protein according to (11), etc.)expressed in cell membranes of the transformant according to (5) (e.g.,the mouse-derived galanin receptor protein-expressible transformantaccording to (24), etc.) by culturing said transformant and theresulting cell stimulating activities via the galanin receptor proteinare measured and compared;

[0109] (60) a method of screening according to (58) or (59) in which thecell-stimulating activity is an activity which accelerates or inhibitsarachidonic acid liberation, acetylcholine liberation, intracellularCa²⁺ liberation, intracellular cAMP production, intracellular cGMPproduction, inositol phosphate production, cell membrane potentialvariation, phosphorylation of intracellular protein, activation ofc-fos, a decrease in pH, insulin secretion, etc. (especially theactivity which accelerates or inhibits the intracellular cAMP productionor insulin secretion);

[0110] (61) a galanin receptor agonist or antagonist obtained by ascreening methods according to any of (31), (34) (e.g., (32), (35),etc.) and (54) to (60);

[0111] (62) an agent for inhibiting acetylcholine liberation, insulinsecretion, learning behavior or feeling of satiety after a mealcharacterized in containing the galanin receptor agonist according to(61);

[0112] (63) an agent for accelerating acetylcholine liberation, insulinsecretion, behavior of learning or feeling of fulfillment after a mealcharacterized in containing the galanin receptor antagonist according to(61);

[0113] (64) an intelligence tropic agent or a remedy for obesity or fordiabetes characterized in containing the galanin receptor antagonistaccording to (40) (e.g., (41), etc.) or (61);

[0114] (65) a kit for screening according to (37) (e.g., (38), etc.),characterized in comprising a cell containing the galanin receptorprotein according to (1) (e.g., the mouse-derived galanin receptorprotein according to (11), etc.);

[0115] (66) a kit for screening according to (37) (e.g., (38), etc.),characterized in containing the membrane fractions of the cells whichcontain the galanin receptor protein according to (1) (e.g., themouse-derived galanin receptor protein according to (11), etc.);

[0116] (67) a galanin receptor agonist or antagonist obtained by the useof the kit for screening according to (37) (e.g., (38), etc.), (65) or(66);

[0117] (68) an agent for inhibiting acetylcholine liberation, insulinsecretion, learning behavior or feeling of fulfillment after a mealcharacterized in containing the galanin receptor agonist according to(67);

[0118] (69) an agent for accelerating acetylcholine liberation, insulinsecretion, behavior of learning or feeling of fulfillment after a mealcharacterized in containing the galanin receptor antagonist according to(67);

[0119] (70) an intelligence tropic agent or a remedy for obesity or fordiabetes characterized in containing the galanin receptor antagonistaccording to (40) (e.g., (41), etc.) or (69); and

[0120] (71) a method of quantitative determination of the galaninreceptor protein or its salt according to (1) (e.g., the mouse-derivedgalanin receptor protein or its salt according to (11), etc.) or thepartial peptide or its salt according to (14) (e.g., the partial peptideof the mouse-derived galanin receptor protein or its salt according to(15), etc.), characterized in that, the antibody according to (51)(e.g., the antibody according to (52), etc.) is contacted with thegalanin receptor protein or its salt according to (1) (e.g., themouse-derived galanin receptor protein or its salt according to (11),etc.) or the partial peptide or its salt according to (14) (e.g., thepartial peptide of the mouse-derived galanin receptor protein or itssalt according to (15), etc.).

[0121] The present invention furthermore provides the following:

[0122] (72) a method of measuring the physiological activity of galanin,characterized in that, the biological activity of the mouse-derived MIN6cells when the mouse-derived MIN cells (FERM BP-4954) or the cellmembrane fractions thereof are contacted with galanin;

[0123] (73) a method of screening a galanin receptor agonist orantagonist, characterized in that, a comparison is made between thecases where (i) galanin is contacted with the mouse-derived MIN6 cells(FERM BP-4954) or cell membrane fractions thereof and (ii) galanin andthe test compound are contacted with the mouse-derived MIN6 cells (FERMBP-4954) or cell membrane fractions thereof;

[0124] (74) a kit for screening for a galanin receptor agonist orantagonist characterized in containing the mouse-derived MIN6 cells(FERM BP-4954) or cell membrane fractions thereof;

[0125] (75) a galanin receptor agonist or antagonist obtained by themethod for screening according to (73) or by the kit for screeningaccording to (74);

[0126] (76) an inhibitor for liberation of acetylcholine, an inhibitorfor secretion of insulin, an inhibitor for the behavior of learning oran inhibitor for feeling satiety after a meal characterized incontaining the galanin receptor agonist according to (75);

[0127] (77) an accelerator for liberation of acetylcholine, anaccelerator for secretion of insulin, an accelerator for the behavior oflearning or an accelerator for feeling satiety after a mealcharacterized in containing the galanin receptor antagonist according to(75);

[0128] (78) a method for screening a galanin receptor agonist orantagonist, characterized in that, the labeled galanin is contacted withthe mouse-derived MIN6 cells (FERM BP-4954) and the labeled galanin andthe test compound are contacted with the mouse-derived MIN6 cells (FERMBP-4954) and the binding amounts of the labeled galanin with saidmouse-derived galanin MIN6 cells in both cases are measured andcompared;

[0129] (79) a method of screening a galanin receptor agonist orantagonist, characterized in that, the labeled galanin is contacted withthe cell membrane fractions of the mouse-derived MIN6 cells (FERMBP-4954) and the labeled galanin and the test compound are contactedwith the cell membrane fractions of the mouse-derived MIN6 cells (FERMBP-4954) and the binding amounts of the labeled galanin with saidmembrane fractions of the mouse-derived MIN6 cells in both cases aremeasured and compared;

[0130] (80) a method of screening a galanin receptor agonist orantagonist, characterized in that, galanin is contacted with themouse-derived MIN6 cells (FERM BP-4954) and galanin and the testcompound are contacted with the mouse-derived MIN6 cells (FERM BP-4954)and the resulting cell-stimulating activities via the mouse-derivedgalanin receptor (especially the activity of secretion of insulin fromMIN6 cells or the activity of inhibiting or accelerating the CAMPproduction in the MIN6 cells) in both cases are measured and compared;

[0131] (81) a method of screening according to the above (80) in whichthe cell-stimulating activity is an activity for accelerating orinhibiting the arachidonic acid liberation, acetylcholine liberation,intracellular Ca²⁺ liberation, intracellular cAMP production,intracellular cGMP production, inositol phosphate production, cellmembrane potential variation, phosphorylation of intracellular protein,activation of c-fos, a decrease in pH, secretion of insulin, etc.(especially the activity which accelerates or inhibits the intracellularcAMP production or the insulin secretion);

[0132] (82) a galanin receptor agonist or antagonist obtained by amethod of screening according to any of (73) and (78) to (81);

[0133] (83) an inhibitor for liberation of acetylcholine, an inhibitorfor secretion of insulin, an inhibitor for the behavior of learning andan inhibitor for feeling fulfillment after a meal characterized incontaining the galanin receptor agonist according to (75) or (82);

[0134] (84) an accelerator for liberation of acetylcholine, anaccelerator for secretion of insulin, an accelerator for the behavior oflearning and an accelerator for feeling satiety after a mealcharacterized in containing the galanin receptor antagonist according to(75) or (82); and

[0135] (85) an intelligence tropic agent or a remedy for obesity or fordiabetes characterized in containing the galanin receptor antagonistaccording to (75) or (82).

[0136] Yet another aspect of the present invention is:

[0137] (86) a partial peptide according to (16) in which the partialpeptide is a region exposed outside the cell membrane of the humangalanin receptor protein molecule according to (13);

[0138] (87) a vector according to (23) in which the vector is anexpression vector for the human galanin receptor protein as indicated bypTS863;

[0139] (88) a transformant according to (25) in which the host cell is aCHO cell;

[0140] (89) a CHO cell according to (88) in which the CHO cell isCHO/pTS863-5 or CHO/pTS863-7; `

[0141] (90) a cell or cell membrane fraction thereof according to (30)in which the cell is CHO/pTS863-5 or CHO/pTS863-7;

[0142] (91) a method of screening the galanin receptor agonist orantagonist according to (33), which comprises carrying out a comparisonbetween the cases where (i) galanin is contacted with the human galaninreceptor protein or salt thereof according to (13) or with the partialpeptide or salt thereof according to (16) and (ii) galanin and the testcompound are contacted with the human galanin receptor protein or saltthereof according to (13) or with the partial peptide or salt thereofaccording to (16);

[0143] (92) a method of screening the galanin receptor agonist orantagonist according to (33), which comprises measuring and comparingthe binding amounts of the labeled galanin to said human galaninreceptor protein, partial peptide thereof or salt thereof in the caseswhere (i) the labeled galanin is contacted with the human receptorprotein or salt thereof according to (13) or with the partial peptide orsalt thereof according to (16) and (ii) the labeled galanin and the testcompound are contacted with the human galanin receptor protein or saltthereof according to (13) or with the partial peptide or salt thereofaccording to (16);

[0144] (93) a method of screening the galanin receptor agonist orantagonist according to (33), which comprises carrying out a comparisonbetween the cases where (i) the labeled galanin is contacted with thecell or cell membrane fraction thereof according to (30) and (ii) thelabeled galanin and the test compound are contacted with the cell or thecell membrane fraction thereof according to (30);

[0145] (94) a method of screening the galanin receptor agonist orantagonist according to (33), which comprises measuring and comparingthe the binding amounts of the labeled galanin with said cell or cellmembrane fraction thereof in the cases where (i) the labeled galanin iscontacted with the cell or the cell membrane fraction thereof accordingto (30) and (ii) the labeled galanin and the test compound are contactwith the cell or the cell membrane fraction thereof according to (30);

[0146] (95) a method of screening the galanin receptor agonist orantagonist according to (33), which comprises measuring and comparingcell stimulating activities via the recombinant human galanin receptor(for example, activities which promote or inhibit the opening of K²⁺channel, closing of N type Ca⁺ channel, liberation of arachidonic acid,liberation of acetylcholine, variations in intracellular Ca²⁺concentration, inhibition of intracellular cAMP production, productionof inositol phosphate, cell membrane potential changes, phosphorylationof intracellular protein, activation of c-fos, decrease in pH, cellmigration activity, secretion of hormones, activation of G protein andcell promulgation, etc.) in the cases where (i) galanin is contactedwith the cell or the cell membrane fraction thereof according to (30)and (ii) galanin and the test compound are contacted with the cell orthe cell membrane fraction thereof according to (30);

[0147] (96) a pharmaceutical composition according to (45) forinhibiting liberation of acetylcholine, inhibiting secretion of insulin,stimulating secretion of growth hormones, inhibiting learning behavioror inhibiting satiety;

[0148] (97) a pharmaceutical composition according to (45) which is aprophylactic or therapeutic agent for schizophrenic illness or stomachulcer or is a sedative;

[0149] (98) a pharmaceutical composition according to (48) for promotingthe acetylcholine liberation, inhibiting the growth hormone secretion,promoting the insulin secretion, promoting the learning behavior orpromoting satiety;

[0150] (99) a pharmaceutical composition according to (48) which is aprophylactic and therapeutic agent for diabetes, Alzheimer's disease ordementia;

[0151] (100) a preventive and therapeutic agent containing the DNAaccording to (18) for a galanin receptor protein-deficient disease; and

[0152] (101) a preventive and therapeutic agent according to (100) inwhich the galanin receptor protein-deficient disease is diabetes,Alzheimer's disease or dementia.

[0153] Yet another aspect of the present invention is:

[0154] (102) a galanin receptor protein according to the above (1) whichcomprises

[0155] an amino acid sequence selected from the group consisting of anamino acid sequence represented by SEQ ID NO: 1, amino acid sequenceswherein one or more amino acid residues (preferably from 1 to 30 aminoacid residues, more preferably from 1 to 10 amino acid residues) aredeleted from the amino acid sequence of SEQ ID NO: 1, amino acidsequences wherein one or more amino acid residues (preferably from 1 to30 amino acid residues, more preferably from 1 to 10 amino acidresidues) are added to the amino acid sequence of SEQ ID NO: 1, andamino acid sequences wherein one or more amino acid residues (preferablyfrom 1 to 30 amino acid residues, more preferably from 1 to 10 aminoacid residues) in the amino acid sequence of SEQ ID NO: 1 aresubstituted with one or more other amino acid residues, or a saltthereof;

[0156] (103) a galanin receptor protein according to the above (1) whichcomprises

[0157] an amino acid sequence selected from the group consisting of anamino acid sequence represented by SEQ ID NO: 2, amino acid sequenceswherein one or more amino acid residues (preferably from 1 to 30 aminoacid residues, more preferably from 1 to 10 amino acid residues) aredeleted from the amino acid sequence of SEQ ID NO: 2, amino acidsequences wherein one or more amino acid residues (preferably from 1 to30 amino acid residues, more preferably from 1 to 10 amino acidresidues) are added to the amino acid sequence of SEQ ID NO: 2, andamino acid sequences wherein one or more amino acid residues (preferablyfrom 1 to 30 amino acid residues, more preferably from 1 to 10 aminoacid residues) in the amino acid sequence of SEQ ID NO: 2 aresubstituted with one or more other amino acid residues, or a saltthereof; and

[0158] (104) a galanin receptor protein according to the above (1) whichcomprises

[0159] an amino acid sequence selected from the group consisting of anamino acid sequence represented by SEQ ID NO: 5, amino acid sequenceswherein one or more amino acid residues (preferably from 1 to 30 aminoacid residues, more preferably from 1 to 10 amino acid residues) aredeleted from the amino acid sequence of SEQ ID NO: 5, amino acidsequences wherein one or more amino acid residues (preferably from 1 to30 amino acid residues, more preferably from 1 to 10 amino acidresidues) are added to the amino acid sequence of SEQ ID NO: 5, andamino acid sequences wherein one or more amino acid residues (preferablyfrom 1 to 30 amino acid residues, more preferably from 1 to 10 aminoacid residues) in the amino acid sequence of SEQ ID NO: 5 aresubstituted with one or more other amino acid residues, or a saltthereof.

[0160] Yet another aspect of the present invention is:

[0161] (105) a process according to the above (27), wherein saidtransformant is produced by transforming a host cell, CHO cell, with avector comprising a nucleotide sequence coding for a human-derivedgalanin receptor protein;

[0162] (106) a pharmaceutical composition comprising an effective amountof an agonist according to the above (40) or a salt thereof in admixturewith a pharmaceutically acceptable diluent, carrier or excipient;

[0163] (107) a pharmaceutical composition according to the above (106),which inhibits liberation of acetylcholine, secretion insulin, learningaction, or satiety;

[0164] (108) a pharmaceutical composition comprising an effective amountof an antagonist according to the above (40) or a salt thereof inadmixture with a pharmaceutically acceptable diluent, carrier orexcipient;

[0165] (109) a pharmaceutical composition according to the above (108),which promotes liberation of acetylcholine, secretion insulin, learningaction, or satiety; and

[0166] (110) a transformant CHO cell capable of expressing human-derivedgalanin receptor proteins.

[0167] As used herein the term “substantial equivalent(s)” means thatthe activity of the protein, e.g., nature of the ligand bindingactivity, and physical characteristics are substantially the same.Substitutions, deletions or insertions of amino acids often do notproduce radical changes in the physical and chemical characteristics ofa polypeptide, in which case polypeptides containing the substitution,deletion, or insertion would be considered to be substantiallyequivalent to polypeptides lacking the substitution, deletion, orinsertion. Substantially equivalent substitutes for an amino acid withinthe sequence may be selected from other members of the class to whichthe amino acid belongs. The non-polar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine, The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid

BRIEF DESCRIPTION OF THE DRAWINGS

[0168]FIG. 1 is the partial nucleotide sequence of the novel receptorprotein cDNA clone, p3H2-34, obtained from mouse pancreatic β-cell line,MIN6, by PCR amplification and the amino acid sequence encoded therebywherein the underlined parts correspond to the synthetic primers usedfor the PCR amplification

[0169]FIG. 2 is the hydrophobicity plotting profile, prepared based uponthe amino acid sequence shown in FIG. 1, wherein the axis of ordinaterepresents an index of hydrophobicity, the axis of abscissa representsthe number of amino acids and numerals 3 to 6 represent the presence ofhydrophobic domains.

[0170]FIG. 3 is the partial amino acid sequence encoded by the novelreceptor protein cDNA included in p3H2-34 relative to the partial aminoacid sequence each of human somatostatin receptor subtype 4 protein(JN0605, human somatostatin receptor subtype 2 protein (B41795) andrat-derived ligand unknown receptor protein (A39297), wherein reverseamino acid residues are in agreement.

[0171]FIG. 4 shows the nucleotide sequence of the mouse-derived galaninreceptor protein cDNA clone PMGR20, which has been cloned with, as aprobe, the cDNA insert in p3H2-34 and the amino acid sequence encodedthereby

[0172]FIG. 5 is the hydrophobicity plotting profile, prepared based uponthe amino acid sequence shown in FIG. 4, wherein the axis of ordinaterepresents an index of hydrophobic property, the axis of abscissarepresents the number of amino acids, and numerals 1 to 7 represent thepresence of hydrophobic domains.

[0173]FIG. 6 is the amino acid sequence (MOUSEGALRECE) of themouse-derived galanin receptor protein encoded by pMGR20, relative tothe amino acid sequence (HUMAGALAMI) of the human-derived galaninreceptor protein, wherein reverse amino acid residues are in agreement.

[0174]FIG. 7 is the plotting profile of the binding amounts (PMB) oflabeled galanin to MIN 6 cells against the concentrations of standardporcine galanin, rat galanin, galanin (1-16) partial peptide or galaninantagonist (galantide).

[0175]FIG. 8 is the plotting profile of the amount of insulin secretionfrom MIN 6 cells against the amount of galanin.

[0176]FIG. 9 is the plotting profile of the amount of insulin secretionfrom MIN 6 cells against the amount of galanin.

[0177]FIG. 10 is the slotting profile of the amount of intracellularcAMP in MIN 6 cells against the amount of galanin.

[0178]FIG. 11 is the nucleotide sequence and deduced amino acid sequence(1st to 135th) of the human galanin receptor protein obtained in Example11.

[0179]FIG. 12 is the nucleotide sequence and deduced amino acid sequence(136th to 349th) of the human galanin receptor protein obtained inExample 11.

[0180]FIG. 13 is the construction of expression plasmid, pTS863,containing the human galanin receptor protein cDNA obtained in Example12. The striped region of expression plasmid, pTS863, indicates thehuman galanin receptor protein cDNA. DHFR; dhfr gene and Amp^(r);ampicillin resistant gene.

[0181]FIG. 14 depicts a profile of northern blot analysis of poly(A)⁺RNA from mouse tissues, Neuro-2a, and MIN6 cells with p3H2-34. Poly(A)⁺RNA (5 μg/lane) was denatured by treatment with glyoxal and thenelectrophoresed on a 1.2% agarose gel. The RNAs were transferred onto anitrocellulose filter and hybridized with a ³²P-labeled cDNA insert ofp3H2-34 as a probe. Lanes: 1, Neuro-2a; 2, MIN6; 3, intestinal smoothmuscle; 4, testis; 5, pancreas; 6, kidney; 7, liver; 8, heart; 9, lung;10, spleen; 11, thymus; 12, brain. Arrowheads indicate the sizes of themolecular weight markers.

[0182]FIG. 15 illustrates a comparison of porcine [¹²⁵I]galanin bindingto CHO cells transformed with or without a mouse galanin receptor cDNA.CHO-MGR20 cells transformed with a full-length translation unit or mocktransformed CHO cells were incubated with [¹²⁵I]galanin (100 pM at finalconcentration) at 37° C. for 1 hr in the absence (open column) orpresence (closed column) of unlabeled porcine galanin (1 μM at finalconcentration). The amounts of [¹²⁵I]galanin bound are represented as apercentages of the radioactivity remaining on the cells after washing.Values indicated are mean±S.E.M. in triplicate.

[0183]FIG. 16 is a primary structure comparison of mouse and humangalanin receptors. Identical residues are indicated by the verticalline. Putative membrane spanning domains I-VII are boxed.

[0184]FIG. 17 depicts a profile of Scatchard analysis of [¹²⁵I] galaninbinding to the membranes from CHO cells transformed with the mousegalanin receptor cDNA. Membrane fractions (1 μg of protein) wereincubated with increasing concentrations of porcine [¹²⁵I] galanin for75 min at 25° C. The results shown are from one representativeexperiment performed in triplicate assays. Each symbol represents themean value±S.E.M. The values for K_(d) and B_(MAX) were 45 pM and 5pmol/mg protein, respectively. B, [¹²⁵I] galanin bound (pmol/mgprotein), B/F, bound to free ratio (pmol/mg protein•nM).

[0185]FIG. 18 illustrates a result of competitive experiments on thebinding of porcine [¹²⁵I] galanin to mouse galanin receptor.Competitions to the porcine [¹²⁵I] galanin (100 pM at finalconcentrations) bindings were examined with unlabeled porcine (Δ), rat(), human (▪) galanins, galanin (1-16) (◯), and M15 (▴). Membranefractions (1 μg of protein) were incubated with the ligands for 75 minat 25° C. The amounts of [¹²⁵I] galanin bound were expressed aspercentages against the control. Each symbol represents the meanvalue±S.E.M. of the triplicate assays. IC₅₀ values were 0.25±0.03 nM(porcine galanin), 0.25±0.01 nM (rat galanin), 0.43±0.03 nM (humangalanin), 0.83±0.01 nM (M15), and 3.6±0.04 nM [galanin-(1-16)],respectively.

[0186]FIG. 19 shows a galanin receptor-mediated inhibition offorskolin-stimulated cAMP production. CHO-MGR20 or mock transformed CHOcells were incubated with forskolin (10 μM) and porcine galanin (0.1 μM)at 37° C. for 30 min. The reaction was terminated by extracting thecells with ice-cold ethanol. The amounts of intracellular cAMP werequantitated by EIA. Values indicated are mean±S.E.M. in triplicateassays.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0187] According to the present invention, galanin receptor proteins andpartial peptides thereof or salts thereof; DNAs comprising a DNA codingfor said galanin receptor protein or its partial peptide; vectorscarrying said DNA; transformants harboring said vector; cell membranefractions obtained from said transformant; processes for producing saidreceptor protein or its partial peptide, or a salt thereof; methods formeasuring the physiological actions of galanin using the galaninreceptor protein (including a cell membrane fraction containing thereceptor protein) or a galanin receptor protein-expressing cell(including the transformant); screening methods for a galanin receptoragonist/antagonist using the galanin receptor protein or a galaninreceptor protein-expressing cell (including the transformant); kits forsaid screening; agonists or antagonists, obtained by said screeningmethod; pharmaceutical compositions containing said agonist orantagonist; antibodies against said receptor protein; immunoassays usingsaid receptor protein or said antibody; use of said receptor protein andencoding DNA; etc. may be successfully provided. For example, templateDNAs coding for part or all of the polypeptide sequence of galaninreceptor protein, can be successfully obtained and various DNA sequencesencoding part or all of the polypeptide sequence of galanin receptorprotein can be isolated and characterized. Further, galanin receptorproteins, partial peptides derived from the galanin receptor protein,modified derivatives or analogues thereof, and salts thereof arerecognized, predicted, deduced, produced, expressed, isolated andcharacterized. More specifically, DNA sequences comprising each anucleotide sequence indicated by a SEQ ID NO selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 6 have beenisolated and characterized. Galanin receptor proteins comprising eachpart or all of an amino acid sequence selected from the group consistingof an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 orSEQ ID NO: 5 and its substantial equivalents thereto, or a salt thereof.

[0188] These galanin receptor proteins are those derived from all cellsand tissues (e.g. amygdaloid nucleus, pituitary gland, pancreas, brain(including whole brain, midbrain nigra and other regions), kidney,liver, gonad, thyroid gland, cholecyst, bone marrow, adrenal, skin,muscle, lung, digestive duct, stomach, blood vessel, heart, thymus,spleen, leukocyte, etc.) of warm-blooded animals (e.g. guinea,pig, rat,mouse, swine, sheep, cattle, horse, monkey, human being, rabbit, cat,dog, etc.), and any of galanin receptor proteins as long as theycomprise an amino acid sequence selected from the group consisting of anamino acid sequence represented by SEQ ID NO: 1, an amino acid sequencerepresented by SEQ ID NO: 2, an amino acid sequence represented by SEQID NO: 5, and substantial equivalents to the amino acid sequencerepresented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5. Thesegalanin receptor proteins may include proteins having an amino acidsequence selected from the group consisting of an amino acid sequencerepresented by SEQ ID NO: 1, an amino acid sequence represented by SEQID NO: 2, and an amino acid sequence represented by SEQ ID NO: 5,proteins wherein the amino acid sequence thereof is about 90% to 99.9%homologous to an amino acid sequence represented by SEQ ID NO: 1, anamino acid sequence represented by SEQ ID NO: 2 or an amino acidsequence represented by SEQ ID NO: 5 and the activity thereof issubstantially equivalent to the protein having an amino acid sequencerepresented by SEQ ID NO: 1, an amino acid sequence represented by SEQID NO: 2 or an amino acid sequence represented by SEQ ID NO: 5 and thelike. The substantially equivalent activity may include ligand bindingactivity, signal information transmitting, etc. The term “substantiallyequivalent” or “substantial equivalent” means that the nature of theligand binding activity and the like is equivalent. Therefore, it isallowable that even differences among grades such as ligand bindingaffinity grades and ligand binding activity grades and quantitativefactors such as molecular weights of receptor proteins are present.

[0189] In one embodiment of the present invention, mouse-derived galaninreceptor proteins are those derived from all mouse-derived cells andtissues (e.g. amygdaloid nucleus, pituitary gland, pancreas, brain,kidney, liver, gonad, thyroid gland, cholecyst, bone marrow, lung,digestive duct, blood vessel, heart, thymus, spleen, leukocyte, etc.),and any of proteins as long as they comprise an amino acid sequencerepresented by SEQ ID NO: 1, and substantial equivalents thereto. Themouse-derived galanin receptor proteins may include proteins having anamino acid sequence represented by SEQ ID NO: 1, proteins wherein theamino acid sequence thereof is about 90% to 99.9% homologous to an aminoacid sequence represented by SEQ ID NO: 1 and the activity thereof issubstantially equivalent to the protein having an amino acid sequencerepresented by SEQ ID NO: 1 and the like. The substantially equivalentactivity may include ligand binding activity, signal informationtransmitting, etc. The term “substantially equivalent” or “substantialequivalent” means that the nature of the ligand binding activity and thelike is equivalent. Therefore, it is allowable that even differencesamong grades such as ligand binding affinity grades and ligand bindingactivity grades and quantitative factors such as molecular weights ofreceptor proteins are present.

[0190] In another embodiment of the present invention, mouse-derivedgalanin receptor proteins include mouse pancreatic β-cell line, MIN6(FERM BP-4954)-derived galanin receptor proteins comprising an aminoacid sequence represented by SEQ ID NO: 1, etc. Examples of themouse-derived galanin receptor protein are mouse-derived galaninreceptor proteins having an amino acid sequence represented by SEQ IDNO: 1, proteins wherein one or more amino acid residues (preferably from1 to 30 amino acid residues, more preferably from 1 to 10 amino acidresidues) are deleted from the amino acid sequence of SEQ ID NO: 1,proteins wherein one or more amino acid residues (preferably from 1 to30 amino acid residues, more preferably from 1 to 10 amino acidresidues) are added to the amino acid sequence of SEQ ID NO: 1, proteinswherein one or more amino acid residues (preferably from 1 to 30 aminoacid residues, more preferably from 1 to 10 amino acid residues) in theamino acid sequence of SEQ ID NO: 1, are substituted with one or moreamino acid residues, etc.

[0191] More specific examples of the mouse-derived galanin receptorprotein are mouse pancreatic β-cell line, MIN6-derived galanin receptorproteins having an amino acid sequence represented by SEQ ID NO: 2,proteins having a substantial amino acid sequence thereto (for example,the amino acid sequence thereof is about 90% to 99.9% homologous to anamino acid sequence represented by SEQ ID NO: 2), proteins wherein oneor more amino acid residues (preferably from 1 to 30 amino acidresidues, more preferably from 1 to 10 amino acid residues) are deletedfrom the amino acid sequence of SEQ ID NO: 2, proteins wherein one ormore amino acid residues (preferably from 1 to 30 amino acid residues,more preferably from 1 to 10 amino acid residues) are added to the aminoacid sequence of SEQ ID NO: 2, proteins wherein one or more amino acidresidues (preferably from 1 to 30 amino acid residues, more preferablyfrom 1 to 10 amino acid residues) in the amino acid sequence of SEQ IDNO: 2, are substituted with one or more amino acid residues, etc.

[0192] In yet another embodiment of the present invention, human galaninreceptor proteins are those derived from all human-derived cells andtissues (e.g. stomach, pituitary gland, pancreas, brain (including wholebrain, midbrain nigra and other regions), kidney, liver, gonad, thyroidgland, cholecyst, bone marrow, adrenal, skin, muscle, lung, digestiveduct, blood vessel, heart, etc.), and any of proteins as long as theycomprise an amino acid sequence represented by SEQ ID NO: 5, andsubstantial equivalents thereto. The human galanin receptor proteins mayinclude proteins having an amino acid sequence represented by SEQ ID NO:5, proteins wherein the amino acid sequence thereof is about 90% to99.9% homologous to an amino acid sequence represented by SEQ ID NO: 5and the activity thereof is substantially equivalent to the proteinhaving an amino acid sequence represented by SEQ ID NO: 5 and the like.The substantially equivalent activity may include ligand bindingactivity, signal information transmitting, etc. The term “substantiallyequivalent” or “substantial equivalent” means that the nature of theligand binding activity and the like is equivalent. Therefore, it isallowable that even differences among grades such as ligand bindingaffinity grades and ligand binding activity grades and quantitativefactors such as molecular weights of receptor proteins are present.

[0193] In another embodiment of the present invention, human galaninreceptor proteins include human-derived galanin receptor proteinscomprising an amino acid sequence represented by SEQ ID NO: 5, etc.Examples of the human galanin receptor protein are human-derived galaninreceptor proteins having an amino acid sequence represented by SEQ IDNO: 5, proteins wherein one or more amino acid residues (preferably from1 to 20 amino acid residues, more preferably from 1 to 10 amino acidresidues) are deleted from the amino acid sequence of SEQ ID NO: 5,proteins wherein one or more amino acid residues (preferably from 1 to20 amino acid residues, more preferably from 1 to 10 amino acidresidues) are added to the amino acid sequence of SEQ ID NO: 5, proteinswherein one or more amino acid residues (preferably from 1 to 20 aminoacid residues, more preferably from 1 to 10 amino acid residues) in theamino acid sequence of SEQ ID NO: 5, are substituted with one or moreamino acid residues, etc.

[0194] A portion of the amino acid sequence may be modified (e.g.addition, deletion, substitution with other amino acids, etc.) in thegalanin receptor proteins of the present invention.

[0195] Furthermore, the galanin receptor proteins of the presentinvention includes those wherein N-terminal Met is protected with aprotecting group (e.g., C₁₋₆ acyl group such as formyl, acetyl, etc.),those wherein the N-terminal side of Glu is cleaved in vivo to make saidGlu pyroglutaminated, those wherein the intramolecular side chain ofamino acids is protected with a suitable protecting group (e.g., C₁₋₆acyl group such as formyl, acetyl, etc.), conjugated proteins such asso-called “glycoproteins” wherein saccharide chains are bonded, etc.

[0196] However, the known human galanin receptor protein having an aminoacid sequence in which the fifteenth Trp in the amino acid sequencerepresented by SEQ ID NO: 5 is substituted with Cys is excluded from thecoverage of the human galanin receptor protein of the present invention.

[0197] The salt of said galanin receptor protein of the presentinvention includes preferably physiologically acceptable acid additionsalts. Examples of such salts are salts thereof with inorganic acids(e.g. hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuricacid, etc.), salts thereof with organic acids (e.g. acetic acid, formicacid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaricacid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid, benzenesulfonic acid, etc.), etc.

[0198] The galanin receptor protein or its salt of the present inventionmay be manufactured from the tissues or cells of warm-blooded animals bypurifying methods which are known per se by those skilled in the art ormethods similar thereto or may be manufactured by culturing thetransformant (or transfectant) (as described herein below) containinggalanin receptor protein encoding DNA. The protein or its salt of thepresent invention may be manufactured by the peptide synthesis asdescribed herein below.

[0199] The galanin receptor protein fragment (the partial peptide ofsaid galanin receptor protein) may include, for example, the site whichis exposed outside cell membranes, among the galanin receptor proteinmolecule. Examples of the partial peptide are peptides containing aregion which is analyzed as an extracellular area (hydrophilic region orsite) in a hydrophobic plotting analysis on the galanin receptorprotein. A peptide which partly contains a hydrophobic region or sitemay be used as well. Further, a peptide which separately contains eachdomain may be used too although the partial peptide (or peptidefragment) which contains plural domains at the same time will be used aswell.

[0200] In an embodiment of the present invention, the partial peptide ofsaid mouse-derived galanin receptor protein may include, for example,the site which is exposed outside cell membranes, among the galaninreceptor protein molecule. Examples of the mouse-derived galaninreceptor partial peptide are peptides containing a region which isanalyzed as an extracellular area (hydrophilic region or site) in ahydrophobic plotting analysis on the galanin receptor protein,represented by FIG. 2.

[0201] The salt of said galanin receptor partial peptide includespreferably physiologically acceptable acid addition salts. Examples ofsuch salts are salts thereof with inorganic acids (e.g. hydrochloricacid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), saltsthereof with organic acids (e.g. acetic acid, formic acid, propionicacid, fumaric acid, maleic acid, succinic acid, tartaric acid, citricacid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid,benzenesulfonic acid, etc.), etc.

[0202] The partial peptide of the galanin receptor protein may bemanufactured by synthesizing methods for peptides which are known per seby those skilled in the art or methods similar thereto or by cleaving(digesting) galanin receptor proteins by a suitable peptidase. Methodsof synthesizing peptide may be any of a solid phase synthesis and aliquid phase synthesis. Thus, a partial peptide (peptide fragment) oramino acids which can construct the protein of the present invention iscondensed with the residual part thereof and, when the product has aprotective group, said protective group is detached whereupon a desiredpeptide can be manufactured. Examples of the known methods forcondensation and for detachment of protective groups include thefollowing {circle over (1)} to {circle over (5)}:

[0203] {circle over (1)} M. Bodanszky and M. A. Ondetti: PeptideSynthesis, Interscience Publishers, New York (1966).

[0204] {circle over (2)} Schroeder and Luebke: The Peptide, AcademicPress, New York, (1965).

[0205] {circle over (3)} Nobuo Izumiya et al.: Fundamentals andExperiments of the Peptide Synthesis, Maruzen K K, Japan (1975).

[0206] {circle over (4)} Haruaki Yajima and Shumpei Sakakibara:“Seikagaku Jikken Koza 1” (Experiments of Biochemistry, Part 1),“Tanpakusitu No Kagaku IV” (Chemistry of Protein, IV), p.205 (1977),Japan.

[0207] {circle over (5)} Haruaki Yajima (ed): Development ofPharmaceuticals (Second Series), Vol. 14, Peptide Synthesis, HirokawaShoten, Japan.

[0208] After the reaction, conventional purifying techniques such assalting-out, extraction with solvents, distillation, columnchromatography, liquid chromatography, electrophoresis,recrystallization, etc. are optionally combined so that the protein ofthe present invention can be purified and isolated. When the proteinobtained as such is a free compound, it may be converted to a suitablesalt by known methods while, when it is obtained as a salt, the salt maybe converted to a free compound or other salt compounds by knownmethods.

[0209] Furthermore, the product may be manufactured by culturing thetransformant (transfectant) containing the DNA coding for said partialpeptide.

[0210] The galanin receptor protein-encoding DNA of the presentinvention may be any coding DNA as long as it contains a nucleotidesequence coding for a galanin receptor protein which contains an aminoacid sequence substantially equivalent to the amino acid sequence havingSEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5 and/or which has an activitysubstantially equivalent to the amino acid sequence having SEQ ID NO: 1,SEQ ID NO: 2 or SEQ ID NO: 5, provided that the known galanin receptorprotein wherein 15th Trp in the the amino acid sequence of SEQ ID NO: 5is replaced with Cys is excluded.

[0211] The DNA of the present invention may be any one of a genome DNA,a genome DNA library, a tissue and cell-derived cDNA, a tissue andcell-derived cDNA library and a synthetic DNA. The vector used for thelibrary may include bacteriophage, plasmid, cosmid, phagemid, etc. TheDNA can be further amplified directly by the reverse transcriptasepolymerase chain reaction (hereinafter briefly referred to as “RT-PCR”)using mRNA fractions prepared from tissues and cells.

[0212] In an embodiment, the DNA coding for the mouse-derived galaninreceptor protein may be any coding DNA as long as it contains anucleotide sequence coding for a mouse-derived galanin receptor proteinwhich contains an amino acid sequence substantially equivalent to theamino acid sequence having SEQ ID NO: 1 and/or which has an activitysubstantially equivalent to the amino acid sequence having SEQ ID NO: 1.Examples of the DNA coding for the mouse-derived galanin receptorprotein comprising the amino acid sequence of SEQ ID NO: 1 includes DNAhaving a nucleotide sequence represented by SEQ ID NO: 3, etc. The DNAcoding for the mouse-derived galanin receptor protein comprising theamino acid sequence of SEQ ID NO: 2 includes DNA having a nucleotidesequence represented by SEQ ID NO: 4, etc.

[0213] In another embodiment, the DNA coding for the human galaninreceptor protein may be any coding DNA as long as it contains anucleotide sequence coding for a human- derived galanin receptor proteinwhich contains an amino acid sequence substantially equivalent to theamino acid sequence having SEQ ID NO: 5 and/or which has an activitysubstantially equivalent to the amino acid sequence having SEQ ID NO: 5,provided that the known human galanin receptor protein wherein 15th Trpin the the amino acid sequence of SEQ ID NO: 5 is replaced with Cys isexcluded. Examples of the DNA coding for the human galanin receptorprotein comprising the amino acid sequence of SEQ ID NO: 5 includes DNAhaving a nucleotide sequence represented by SEQ ID NO: 6, etc.

[0214] The DNA completely coding for the galanin receptor protein of thepresent invention can be cloned by

[0215] (1) carrying out the PCR amplification using a synthetic DNAprimer having a partial nucleotide sequence (nucleotide fragment) of thegalanin receptor protein; or

[0216] (2) effecting the selection of a DNA constructed in a suitablevector, based on the hybridization with a labeled DNA fragment havingpart or all of the region encoding a galanin receptor protein (e.g.,human galanin receptor protein, etc.) or a labeled synthetic DNA havingpart or all of the coding region thereof. The hybridization is carriedout according to methods as disclosed in, for example, MolecularCloning, 2nd Ed., J. Sambrook et al., Cold Spring Harbor Lab. Press,1989. When a DNA library commercially available in the market is used,the hybridization is carried out according to protocols or manualsattached thereto.

[0217] For example, the DNA completely coding for the mouse-derivedgalanin receptor protein of the present invention is cloned by (1)carrying out the PCR amplification using a synthetic DNA primer having apartial nucleotide sequence (nucleotide fragment) of the mouse-derivedgalanin receptor protein; or (2) effecting the selection of a DNAconstructed in a suitable vector, based on the hybridization with alabeled DNA fragment having part or all of the region encoding a humanor mouse-derived galanin receptor protein or a labeled synthetic DNAhaving part or all of the coding region thereof.

[0218] The cloned galanin receptor protein-encoding DNA of the presentinvention can be used as it is, or can be used, as desired, aftermodifications including digestion with a restriction enzyme or additionof a linker or adapter, etc. depending upon objects. The DNA may have aninitiation codon, ATG, on the 5′ terminal side and a termination codon,TAA, TGA or TAG, on the 3′ terminal side. These initiation andtermination codons can be ligated by using a suitable synthetic DNAadapter.

[0219] A vector containing the galanin receptor protein-encoding DNA(for example, an expression vector for the galanin receptor protein;specifically, an expression plasmid comprising the human galaninreceptor protein-encoding DNA, etc.) can be produced by, for example,(a) cutting out a target DNA fragment from the galanin receptorprotein-encoding DNA of the present invention and (b) ligating thetarget DNA fragment with the downstream site of a promoter in a suitableexpression vector (for example, an expression plasmid compatible withthe human galanin receptor protein-encoding DNA, etc.).

[0220] The vector may include plasmids derived from Escherichia coli(e.g., pBR322, pBR325, pUC12, pUC13, etc.), plasmids derived fromBacillus subtilis (e.g., pUB110, pTP5, pC194, etc.), plasmids derivedfrom yeasts (e.g., pSH19, pSH15, etc.), bacteriophages such as λ-phage,and animal virus such as retrovirus, vaccinia virus and baculovirus.

[0221] According to the present invention, any promoter can be used aslong as it is compatible with a host which is used for expressing agene. When the host for the transformation is E. coli, the promoters arepreferably trp promoters, lac promoters, recA promoters, λ_(PL)promoters, lpp promoters, etc. When the host for the transformation isthe Bacillus, the promoters are preferably SPO1 promoters, SPO2promoters, penP promoters, etc. When the host is an yeast, the promotersare preferably PHO5 promoters, PGK promoters, GAP promoters, ADHpromoters, etc. When the host is an animal cell, the promoters includeSV40-derived promoters, retrovirus promoters, metallothionein promoters,heat shock promoters, cytomegalovirus (CMV) promoters, SRα promoters,etc. An enhancer can be effectively utilized for the expression.

[0222] As required, furthermore, a host-compatible signal sequence isadded to the N-terminal side of the galanin receptor protein. When thehost is E. coli, the utilizable signal sequences may include alkalinephosphatase signal sequences, OmpA signal sequences, etc. When the hostis the Bacillus, they may include α-amylase signal sequences, subtilisinsignal sequences, etc. When the host is an yeast, they may includemating factor α signal sequences, invertase signal sequences, etc. Whenthe host is an animal cell, they may include insulin signal sequences,α-interferon signal sequences, antibody molecule signal sequences, etc.

[0223] Further, a preferred method of constructing an expression plasmidamong the vectors containing the human galanin receptor protein DNA ofthe present invention will be concretely given as hereunder.

[0224] Examples of the plasmid used are pAKKO-111 (sometimes referred toas pA1-11), pRc/CMV, pRc/RSV, etc. and, among them, the use of pAKKO-111(pA1-11) is preferred. With respect to a promoter, anything may be usedso far as it functions in an effective manner in host cells and itsexamples are SV40 early gene promoter, CMV promoter, HSV-TK promoter,SRα promoter, RSV promoter, etc. Among them, CMV promoter and SRαpromoter are preferred and the use of SRα promoter is particularlypreferred.

[0225] With respect to an expression plasmid, the use of the agentcontaining an enhancer, a splicing signal, a poly A adding signal, aselective marker, etc. besides the above-mentioned ones is preferred.Examples of the selective marker are dihydrofolate reductase(hereinafter, sometimes referred to as “dhfr”) gene and neomycinphosphate transferase (hereinafter, sometimes referred to as “neo”gene). The dhfr gene gives a resistance to methotrexate,(MTX) while theneo gene gives a resistance to G-418. Especially when a dhfrgene-deficient CHO cell is used and a dhfr gene is utilized as aselective marker, it is possible to select even by a medium free fromthymidine.

[0226] Specific and preferred examples of the expression plasmidcarrying the human galanin receptor protein encoding DNA of the presentinvention are those in which the above-mentioned promoters (e.g.,particularly, SRα-promoter, etc.) are inserted in the upstream of thehuman galanin receptor protein DNA and, preferably, an SV early genepoly A addition signal is inserted to the downstream of the humangalanin receptor protein DNA followed by inserting dhfr gene,ampicillin-resisting gene, etc. into the downstream of the poly Aaddition signal.

[0227] More specific and preferred example is an expression plasmiddesignated pTS863 (FIG. 13) in which SRα promoter is inserted in anupstream of the human galanin receptor protein DNA, an SV early genepoly A addition signal is inserted in a downstream of the human galaninreceptor protein DNA, a dhfr gene is inserted in a downstream thereofand then an ampicillin-resisting gene is inserted in the downstreamthereof, etc.

[0228] When the expression plasmid containing the human galanin receptorprotein DNA prepared as such is introduced into a host cell, it ispossible to produce a cell which is able to highly express the DNA whichcodes for the human galanin receptor protein.

[0229] A transformant or transfectant is produced by using the vectorthus constructed, which carries the galanin receptor protein-encodingDNA of the present invention. The host may be, for example, Escherichiamicroorganisms, Bacillus microorganisms, yeasts, insect cells, animalcells, etc. Examples of the Escherichia and Bacillus microorganismsinclude Escherichia coli K12-DH1 [Proc. Natl. Acad. Sci. USA, Vol. 60,160 (1968)], JM103 [Nucleic Acids Research, Vol. 9, 309 (1981)], JA221[Journal of Molecular Biology, Vol. 120, 517 (1978)], HB101 [Journal ofMolecular Biology, Vol. 41, 459 (1969)], C600 [Genetics, Vol. 39, 440(1954)], etc. Examples of the Bacillus microorganism are, for example,Bacillus subtilis MI114 [Gene, Vol. 24, 255 (1983)], 207-21 [Journal ofBiochemistry, Vol. 95, 87 (1984)], etc. The yeast may be, for example,Saccharomyces cerevisiae AH22, AH22R⁻, NA87-11A, DKD-5D, 20B-12, etc.The insect may include a silkworm (Bombyx mori larva), [Maeda et al,Nature, Vol. 315, 592 (1985)] etc. The host animal cell may be, forexample, monkey-derived cell line, COS-7, Vero, Chinese hamster ovarycell line (CHO cell), DHFR gene-deficient Chinese hamster cell line(dhfr⁻ CHO cell), CHO K-1, human FL cell, 293 cell, L cell, myelomacell, C127 cell, Balb/c3T3 cell, Sp-2/0 cell, etc.

[0230] Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. Transformation ofEscherichia microorganisms can be carried out in accordance with methodsas disclosed in, for example, Proc. Natl. Acad. Sci. USA, Vol. 69, 2110(1972), Gene, Vol. 17, 107 (1982), etc. Transformation of Bacillusmicroorganisms can be carried out in accordance with methods asdisclosed in, for example, Molecular & General Genetics, Vol. 168, 111(1979), etc. Transformation of the yeast can be carried out inaccordance with methods as disclosed in, for example, Proc. Natl. Acad.Sci. USA, Vol. 75, 1929 (1978), etc. The insect cells can be transformedin accordance with methods as disclosed in, for example, Bio/Technology,6, 47-55, 1988. The animal cells can be transformed by methods asdisclosed in, for example, Virology, Vol. 52, 456, 1973, etc. Thetransformants or transfectants harboring the expression vector carryinga galanin receptor protein-encoding DNA are produced according to theaforementioned techniques.

[0231] Among the above-mentioned host cells, animal cells areparticularly preferred as the host cell for an expression plasmidcontaining the human galanin receptor protein DNA of the presentinvention. The examples thereof are 293 cell, CHO cell, Vero cell, Lcell, myeloma cell, C127 cell, Balb/c3T3 cell and Sp-2/O cell, etc.Among them, CHO cell and 293 cell are preferred and particularly CHOcell [Journal of Experiment of Medicine, 108, 945(1958)] is morepreferred. Among said CHO cell, the preferred ones are dhfrgene-deficient CHO cell (hereinafter, sometimes referred to as CHO(dhfr⁻) cell) [Proceedings of the National Academy of Sciences of the U.S. A., 77, 4216-4220(1980)], CHO K-1 cell [Proceedings of the NationalAcademy of Sciences of the U. S. A., 60, 1275(1968)], etc. When dhfrgene is inserted in an expression plasmid as a selective marker,CHO(dhfr⁻) and the like are suitable.

[0232] With respect to the combination of the expression plasmid withthe host cell, the preferred one can be suitably selected and, forexample, CHO(dhfr⁻) cell and the like are suitable as the host cell ofthe expression plasmid (FIG. 13) indicated by pTS863. In introducing theexpression plasmid into animal cells, known methods such as a calciumphosphate method [Graham, F. L. and van der Eb, A. J.: Virology, 52,456-467(1973)], an electroporation [Neumann, E. et al., EMBO Journal,1,841-845(1982)], etc. may be used.

[0233] As such, a transformant in which a transformation is carried outusing a vector containing a human galanin receptor protein DNA isproduced. In addition, the transformant prepared by a transformationusing an expression plasmid containing the human galanin receptorprotein DNA may be used for the manufacture of human galanin receptorprotein.

[0234] Cells which are able to highly express the human galanin receptorprotein can be obtained by selecting the cells wherein theabove-mentioned expression plasmid is incorporated in the chromosome bymeans of clone selection. Briefly, the transformant is first selectedusing the above-mentioned selective marker as an index for selection.Then the transformant produced as such using the selective marker isrepeatedly subjected to a clone selection to give a cell strain whichstably exhibits a high ability of expressing the human galanin receptorprotein. When a dhfr gene is used as a selective marker, the resistingcells are selected by a culture with a sequential increase in themethotrexate (MTX) concentration to amplify the introduced gene in thecells whereby a cell strain exhibiting far higher expression can beobtained.

[0235] Even when CHO (dhfr⁻) cell is used as a host, the CHO containingan expression plasmid indicated by pTS863 also has a dhfr gene as aresult because a dhfr gene is introduced, for example, into anexpression plasmid indicated by pTS863 (FIG. 13). In this specification,the CHO cell obtained by giving an expression plasmid (e.g. pTS 863,etc.) containing dhfr gene may be sometimes referred to as “CHO(dhfr⁺)cell”.

[0236] An example of the transformant which is able to highly expressthe human galanin receptor protein DNA in the present invention is a CHO(dhfr⁺) cell obtained by giving an expression plasmid indicated bypTS863 obtained in Example 11 (mentioned herein later) to a CHO (dhfr⁻)cell, etc. More specific examples are CHO (dhfr⁺) cell indicated byCHO/pTS863-5, CHO (dhfr⁺) cell indicated by CHO/pTS863-7, etc. Ascompared with the known human galanin receptor protein-expressing COScells, the above-mentioned CHO (dhfr⁺) cells are able to express moreamount of human galanin receptor protein and, further, there are somewhich exhibit a receptor activity (e.g., ligand binding activity, etc.)of about 10 to 100-fold (preferably about 100-fold) as compared withnatural tissues containing human galanin receptor proteins (e.g. humanmelanoma Bowes cells, etc.). Accordingly, those CHO (dhfr⁺) cells areeffective in conducting a method of screening for the human galaninreceptor agonist/antagonist which will be mentioned herein later.

[0237] The cells which contain the human galanin receptor protein of thepresent invention can be also manufactured by culturing the transformantcontaining the vector (particularly, the expression plasmid) carryingthe human galanin receptor protein DNA of the present invention under acondition where the human galanin receptor protein DNA can be expressed.

[0238] Cultivation of the transformant (transfectant) in which the hostis Escherichia or Bacillus microorganism can be carried out suitably ina liquid culture medium. The culture medium may contains carbon sources,nitrogen sources, minerals, etc. necessary for growing the transformant.The carbon source may include glucose, dextrin, soluble starch, sucrose,etc. The nitrogen source may include organic or inorganic substancessuch as ammonium salts, nitrates, corn steep liquor, peptone, casein,meat extracts, bean-cakes, potato extracts, etc. Examples of theminerals may include calcium chloride, sodium dihydrogen phosphate,magnesium chloride, etc. It is further allowable to add yeasts,vitamins, growth-promoting factors, etc. It is desired that the culturemedium is pH from about 5 to about 8.

[0239] The Escherichia microorganism culture medium is preferably an M9medium containing, for example, glucose and casamino acid (Miller,Journal of Experiments in Molecular Genetics), 431-433, Cold SpringHarbor Laboratory, New York, 1972. Depending on necessity, the mediummay be supplemented with drugs such as 3β-indolyl acrylic acid in orderto improve efficiency of the promoter. In the case of the Escherichiahost, the cultivation is carried out usually at about 15 to 43° C. forabout 3 to 24 hours. As required, aeration and stirring may be applied.In the case of the Bacillus host, the cultivation is carried out usuallyat about 30 to 40° C. for about 6 to 24 hours. As required, aeration andstirring may be also applied. In the case of the transformant in whichthe host is an yeast, the culture medium used may include, for example,a Burkholder minimum medium [Bostian, K. L. et al., Proc. Natl. Acad.Sci. USA, Vol. 77, 4505 (1980)], an SD medium containing 0.5% casaminoacid [Bitter, G. A. et al., Proc. Natl. Acad. Sci. USA, Vol. 81, 5330(1984)], etc. It is preferable that pH of the culture medium is adjustedto be from about 5 to about 8. The cultivation is carried out usually atabout 20 to 35° C. for about 24 to 72 hours. As required, aeration andstirring may be applied. In the case of the transformant in which thehost is an insect, the culture medium used may include those obtained bysuitably adding additives such as passivated (or immobilized) 10% bovineserum and the like to the Grace's insect medium (Grace, T. C. C.,Nature, 195, 788 (1962)). It is preferable that pH of the culture mediumis adjusted to be about 6.2 to 6.4. The cultivation is usually carriedout at about 27° C. for about 3 to 5 days. As desired, aeration andstirring may be applied. In the case of the transformant in which thehost is an animal cell, the culture medium used may include MEM medium[Science, Vol. 122, 501 (1952)], DMEM medium [Virology, Vol. 8, 396(1959)], RPMI 1640 medium [Journal of the American Medical Association,Vol. 199, 519 (1967)], 199 medium [Proceedings of the Society for theBiological Medicine, Vol. 73, 1 (1950)], α-MEM medium, etc. which arecontaining, for example, about 5 to 20% of fetal calf serum. Especiallywhen CHO (dhfr⁻) cells and dhfr selective marker gene are used, it ispreferred to use a DMEM medium containing a dialyzed fetal bovine serumwhich rarely contains thymidine. It is preferable that the pH is fromabout 6 to about 8. The cultivation is usually carried out at about 30to 40° C. for about 15 to 72 hours. As required, medium exchange,aeration and stirring may be applied.

[0240] As such, cells containing the human galanin receptor protein canbe manufactured from the transformant retaining the vector(particularly, expression plasmid) containing the human galanin receptorprotein-encoding DNA. Examples of the cell containing the human galaninreceptor protein are CHO cells containing the human galanin receptorprotein and the like. The cells containing the human galanin receptorprotein can be obtained by culturing CHO (dhfr⁺) cell indicated byCHO/pTS863-5, CHO (dhfr⁺) cell indicated by CHO/pTS863-7, etc.

[0241] Separation and purification of the galanin receptor protein (forexample, human galanin receptor protein, etc.) from the above-mentionedcultures can be carried out according to methods described herein below.

[0242] To extract galanin receptor proteins from the culturedmicroorganisms or cells, the microorganisms or cells are collected byknown methods after the cultivation, suspended in a suitable buffersolution, disrupted by ultrasonic waves, lysozyme and/or freezing andthawing, etc. and, then, a crude extract of the galanin receptor proteinis obtained by centrifugation or filtration. Other conventionalextracting or isolating methods can be applied. The buffer solution maycontain a protein-denaturing agent such as urea or guanidinehydrochloride or a surfactant such as Triton X-100 (registeredtrademark, hereinafter often referred to as “TM”).

[0243] In the case where galanin receptor proteins are secreted intoculture media, supernatant liquids are separated from the microorganismsor cells after the cultivation is finished and the resulting supernatantliquid is collected by widely known methods. The culture supernatantliquid and extract containing galanin receptor proteins can be purifiedby suitable combinations of widely known methods for separation,isolation and purification. The widely known methods of separation,isolation and purification may include methods which utilizessolubility, such as salting out or sedimentation with solvents methodswhich utilizes chiefly a difference in the molecular size or weight,such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamidegel electrophoresis, methods utilizing a difference in the electriccharge, such as ion-exchange chromatography, methods utilizing specificaffinity such as affinity chromatography, methods utilizing a differencein the hydrophobic property, such as inverse-phase high-performanceliquid chromatography, and methods utilizing a difference in theisoelectric point such as isoelectric electrophoresis, etc.

[0244] In case where the galanin receptor protein thus obtained is in afree form, the free protein can be converted into a salt thereof byknown methods or method analogous thereto. In case where the galaninreceptor protein thus obtained is in a salt form vice versa, the proteinsalt can be converted into a free form or into any other salt thereof byknown methods or method analogous thereto.

[0245] The galanin receptor protein produced by the transformant can bearbitrarily modified or a polypeptide can be partly removed therefrom,by the action of a suitable protein-modifying enzyme before or after thepurification. The protein-modifying enzyme may include trypsin,chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase, etc.The activity of the galanin receptor protein thus formed can be measuredby experimenting the coupling (or binding) with a ligand includinggalanin or by enzyme immunoassays (enzyme linked immunoassays) usingspecific antibodies.

[0246] When the DNA which codes for the known human galanin receptorprotein is used in the above-mentioned means instead of that which codesfor the human galanin receptor protein of the present invention, it isalso possible to isolate the cells which highly express recombinanthuman galanin receptor protein or to isolate recombinant human galaninreceptor protein.

[0247] Although the known human galanin receptor protein is expressed byCOS cells, the expressed amount in the case of the COS cells are usuallysmall. However, in accordance with a method of constructing theexpression plasmid of the present invention, it is possible tomanufacture not only the cells (particularly CHO cells) which highlyexpress the human galanin receptor protein of the present invention butalso the cells (particularly CHO cells) which highly express the knownhuman galanin receptor protein.

[0248] The cell membrane fraction of a cell containing said galaninreceptor protein (for example, cell membrane fraction of a cellcontaining human galanin receptor protein, cell membrane fraction of acell containing mouse-derived galanin receptor protein, etc.) is a cellmembrane-rich fraction which is prepared by methods per se known tothose of skill in the art or methods similar thereto after disruption ofcells containing the galanin receptor protein (for example, the humangalanin receptor protein, the mouse-derived galanin receptor protein,etc.). Examples of cell disruption may include a method for squeezingcells using a Potter-Elvejem homogenizer, a disruption by a Waringblender or a Polytron (manufactured by Kinematica), a disruption byultrasonic waves, a disruption via blowing out cells from small nozzlestogether with applying a pressure using a French press or the like, etc.In the fractionation of the cell membrane, a fractionation method bymeans of centrifugal force such as a fractional centrifugal separationand a density gradient centrifugal separation is mainly used. Forexample, disrupted cellular liquid is centrifuged at a low speed (500rpm to 3,000 rpm) for a short period (usually, from about one to tenminutes), the supernatant liquid is further centrifuged at a high speed(15,000 rpm to 30,000 rpm) usually for 30 minutes to two hours and theresulting precipitate is used as a membrane fraction. Said membranefraction contains a lot of the expressed galanin receptor protein (forexample, the human galanin receptor protein, the mouse-derived galaninreceptor protein, etc.) and a lot of membrane components such asphospholipids and membrane proteins derived from the cells.

[0249] The amount of the galanin receptor protein in the membranefraction cell containing said galanin receptor protein is preferably 10³to 10⁸ molecules per cell or, suitably, 10⁵ to 10⁷ molecules per cell.Incidentally, the more the expressed amount, the higher the ligandbinding activity (specific activity) per membrane fraction whereby theconstruction of a highly sensitive screening system becomes possibleand, moreover, it may enable us to measure a large amount of sampleswithin the same lot.

[0250] The galanin receptor protein, the partial peptide thereof and thegalanin receptor protein-encoding DNA of the present invention can beused for:

[0251] {circle over (1)} obtaining an antibody and an antiserum,

[0252] {circle over (2)} constructing a system for expressing arecombinant receptor protein,

[0253] {circle over (3)} developing a receptor-binding assay systemusing the above developing system and screening pharmaceutical candidatecompounds,

[0254] {circle over (4)} designing drugs based upon the comparison withligands and receptors which have a similar or analogous structure,

[0255] {circle over (5)} preparing a probe in the analysis of genes andpreparing a PCR primer,

[0256] {circle over (6)} gene manipulating therapy,

[0257] {circle over (7)} producing a transgenic animal (for example,transgenic mouse, etc.),

[0258] {circle over (8)} producing a model animal suffering fromdiseases caused by gene deficiency, etc.

[0259] In particular, it is allowable to screen a galanin receptoragonist or antagonist specific to a warm-blooded animal such as humanbeing by a receptor-binding assay system which uses a system forexpressing a recombinant galanin receptor protein of the presentinvention. The agonist or antagonist thus screened or characterizedpermits various applications including prevention and/or therapy of avariety of diseases.

[0260] For example, the mouse-derived galanin receptor protein, thepartial peptide of the mouse-derived galanin receptor protein and theDNA which codes for the mouse-derived galanin receptor protein can beused for {circle over (1)} obtaining antibody and antiserum; {circleover (2)} construction of an expression system of the recombinantreceptor protein; {circle over (3)} development of the receptor-bindingassay system using said expression system and screening of the candidatecompounds as pharmaceuticals; {circle over (4)} conducting a drug designbased upon a comparison with ligands and receptors which have a similaror analogous structure; {circle over (5)} preparing probes and designingPCR primers in gene diagnosis; {circle over (6)} gene therapy, etc.

[0261] The human galanin receptor protein, the partial peptide of thehuman galanin receptor protein and the DNA which codes for the humangalanin receptor protein can be used for {circle over (1)} obtainingantibody and antiserum; {circle over (2)} construction of an expressionsystem of the recombinant receptor protein; {circle over (3)}development of the receptor-binding assay system using said expressionsystem and screening of the candidate compounds as pharmaceuticals;{circle over (4)} conducting a drug design based upon a comparison withligands and receptors which have a similar or analogous structure;{circle over (5)} preparing probes and designing PCR primers in genediagnosis; {circle over (6)} gene therapy, etc. Especially when thereceptor binding assay system utilizing the expression system for thehuman galanin receptor protein of the present invention is used, it ispossible to screen the galanin receptor agonist or antagonist which isspecific to warm-blooded animals (especially, human being) whereuponsaid agonist or antagonist can be used as a preventive and therapeuticagent for various diseases.

[0262] Concretely described below are uses of galanin receptor proteins,partial peptides thereof (peptide fragments thereof), galanin receptorprotein-encoding DNAs and antibodies against the galanin receptorprotein according to the present invention.

[0263] (1) Quantitative Measurement of Galanin

[0264] The galanin receptor protein, a partial peptide thereof or a saltthereof has a binding property to galanin and, therefore, it is capableof determining quantitatively an amount of galanin in vivo with goodsensitivity.

[0265] This quantitative measurement may be carried out by, for example,combining with a competitive method. Thus, samples to be measured iscontacted with galanin receptor proteins or partial peptide thereof sothat the galanin concentration in said sample can be measured. In oneembodiment of the quantitative measurement, the protocols described inthe following {circle over (1)} and {circle over (2)} or the methodssimilar thereto may be used:

[0266] {circle over (1)} Hiroshi Irie (ed): “Radioimmunoassay”(Kodansha, Japan, 1974); and

[0267] {circle over (2)} Hiroshi Irie (ed): “Radioimmunoassay, SecondSeries” (Kodansha, Japan, 1979).

[0268] Further, the quantitative determination method of galaninaccording to the present invention can be used as a diagnostic methodfor the diseases caused by increase/decrease in galanin concentrationssuch as stomach ulcer, diabetes and Alzheimer's disease.

[0269] (2) Screening of Galanin Receptor Agonist and/or Antagonist

[0270] Galanin receptor proteins or partial peptides thereof are used.Alternatively, expression systems for recombinant type galanin receptorproteins or partial peptides thereof are constructed and receptorbinding assay systems using said expression system are used. In theseassay systems, it is possible to screen compounds (e.g. peptides,proteins, nonpeptidic compounds, synthetic compounds, fermentedproducts, cell extracts, plant extracts, animal tissue extracts, etc.)or salts thereof which inhibits the binding of galanin with the galaninreceptor protein. Such a compound includes a compound exhibiting agalanin receptor-mediated cell stimulating activity (e.g. activity ofpromoting or activity of inhibiting physiological reactions includingliberation of arachidonic acid, liberation of acetylcholine,endocellular Ca²⁺ liberation, endocellular cAMP production, endocellularcGMP production, production of inositol phosphate, changes in cellmembrane potential, phosphorylation of endocellular proteins, activationof c-fos, lowering of pH, secretion of insulin, etc.; in particular,activity of promoting or activity of inhibiting endocellular cAMPproduction and secretion of insulin) (so-called “galanin receptoragonist”), a compound free of such a cell stimulating activity(so-called “galanin receptor antagonist”), etc.

[0271] Thus, the present invention provides a method of screening agalanin receptor agonist and/or galanin receptor antagonist with agalanin receptor protein or a salt thereof, characterized in comparingthe following cases:

[0272] (i) the case wherein galanin is contacted with the galaninreceptor protein or salt thereof, or a partial peptide thereof or a saltthereof; and

[0273] (ii) the case wherein galanin is contacted with a mixture of thegalanin receptor protein or salt thereof or the partial peptide or saltthereof and said test sample (including a test compound).

[0274] In said screening method, one characteristic feature of thepresent invention resides in that the amount of galanin bonded with saidgalanin receptor protein (for example, human or mouse-derived galaninreceptor protein) or partial peptide thereof, the cell stimulatingactivity of galanin, etc. are measured in the case where (i) galanin iscontacted with said galanin receptor protein (for example, human ormouse-derived galanin receptor protein) or its partial peptide and inthe case where (ii) galanin and a test sample (including a testcompound) are contacted with the galanin receptor protein or its partialpeptide, respectively and then compared therebetween.

[0275] In the screening of the galanin receptor agonist or antagonist,it may be considered to use human hippocampus as a human galaninreceptor protein source. However, tissues derived from human being arehardly available and, therefore, they are not suitable for use inscreening whereupon recombinant human galanin receptor proteins whichare abundantly expressed in cells (particularly, animal cells such asCHO cells) are suitable in practice. More preferably, the cell strain inwhich human galanin receptor proteins are continuously and stablyexpressed is advantageously used.

[0276] Accordingly, the human galanin receptor protein of the presentinvention or the salt thereof, the human galanin receptorprotein-partial peptide of the present invention or the salt thereof andthe cell or cell fraction thereof containing the human galanin receptorprotein of the present invention are remarkably useful as a reagent forscreening the galanin receptor agonist or antagonist.

[0277] Briefly, the present invention offers a method of screeninggalanin receptor agonist or antagonist, characterized in that, the humangalanin receptor protein of the present invention or the salt thereof,the partial peptide of the human galanin receptor protein of the presentinvention or the salt thereof or the cells or cell fraction thereofcontaining the human galanin receptor protein of the present inventionis used.

[0278] More specifically, the present invention offers:

[0279] (I) a method of screening the human galanin receptor agonist orantagonist, characterized in that, a comparison is conducted between thecases where (i) the human galanin receptor protein of the presentinvention or the partial peptide or the salt thereof is contacted withgalanin and where (ii) the human galanin receptor protein of the presentinvention or the partial peptide or the salt thereof is contacted withgalanin and a test compound; and

[0280] (II) a method of screening the galanin receptor agonist orantagonist, characterized in that, a comparison is conducted between thecases where (i) cells containing the human galanin receptor protein ofthe present invention or cell membrane fraction thereof are contactedwith galanin and where (ii) cells containing the human galanin receptorprotein of the present invention or cell membrane fraction thereof arecontacted with galanin and a test compound.

[0281] To be more specific, in the screening methods (I) and (II) of thepresent invention, the cell-stimulating activity and/or binding amountof galanin to said human galanin receptor protein or partial peptide orsalt thereof or cells containing the human galanin receptor protein orcell membrane fraction thereof in (i) and (ii) are measured andcompared.

[0282] In one more specific embodiment of the present invention,

[0283] {circle over (1)} a method of screening a galanin receptoragonist and/or galanin receptor antagonist or a salt thereof,characterized in that, when a labeled galanin is contacted with agalanin receptor protein (e.g., human-derived galanin receptor protein,etc.) or a partial peptide thereof and when a labeled galanin and a testcompound are contacted with a galanin receptor protein (e.g.,human-derived galanin receptor protein, etc.) or a partial peptidethereof, the amounts of the labeled galanin bonded with said protein orpartial peptide thereof or salt thereof are measured and compared;

[0284] {circle over (2)} (a) (i) a method of screening a galaninreceptor agonist and/or galanin receptor antagonist or a salt thereof,characterized in that, when a labeled galanin is contacted with galaninreceptor protein (e.g., human or mouse galanin receptor protein,etc.)-containing cells (e.g., mouse MIN 6 cell (FERM BP-4954), etc.) ora membrane fraction of said cells and when a labeled galanin and a testcompound are contacted with galanin receptor protein (e.g., human ormouse galanin receptor protein, etc.)-containing cells (e.g., mouse MIN6 cell (FERM BP-4954), etc.) or a membrane fraction of said cells, theamounts of the labeled galanin binding with said protein or partialpeptide thereof or a salt thereof are measured and compared;

[0285] (b) (ii) a method of screening a galanin receptor agonist and/orgalanin receptor antagonist or a salt thereof, characterized in that,when a galanin receptor protein-activating compound (e.g. galanin) iscontacted with galanin receptor protein (e.g., human or mouse galaninreceptor protein, etc.)-containing cells (e.g., mouse MIN 6 cell (FERMBP-4954), etc.) or a membrane fraction of said cells and when thegalanin receptor protein-activating compound and a test compound arecontacted with galanin receptor protein (e.g., human or mouse galaninreceptor protein, etc.)-containing cells (e.g., mouse MIN 6 cell (FERMBP-4954), etc.) or a membrane fraction of said cells, the resultinggalanin receptor protein-mediated cell stimulating activities (e.g.activities of promoting or activities of inhibiting physiologicalresponses including the opening of K⁺ channel, closing of N type Ca⁺channel liberation of arachidonic acid, liberation of acetylcholine,endocellular Ca²⁺ changes, endocellular cAMP production (or itsdepression), endocellular cGMP production, insulin secretion, productionof inositol phosphate, cell membrane potential changes, phosphorylationof endocellular proteins, activation of c-fos, decrease of pH, cellmigration activity, secretion of hormones, activation of G protein, cellpromulgation, etc.) are measured and compared; and

[0286] {circle over (3)} a method of screening a galanin receptoragonist and/or galanin receptor antagonist or a salt thereof,characterized in that, when a labeled galanin is contacted with galaninreceptor proteins (e.g., human or mouse galanin receptor proteins, etc.)expressed on the cell membrane by culturing a transformant containing agalanin receptor protein encoding DNA and when a labeled galanin and atest compound are contacted with galanin receptor proteins (e.g., humanor mouse galanin receptor proteins, etc.) expressed on the cell membraneby culturing a transformant containing a galanin receptor proteinencoding DNA, the amounts of the labeled galanin binding with saidgalanin receptor protein are measured and compared;

[0287] {circle over (4)} a method of screening a galanin receptoragonist and/or galanin receptor antagonist or a salt thereof,characterized in that, when a galanin receptor protein-activatingcompound (e.g. galanin) is contacted with galanin receptor proteins(e.g., human or mouse galanin receptor proteins, etc.) expressed on cellmembranes by culturing transformants containing galanin receptorprotein-encoding DNA and when a galanin receptor protein-activatingcompound and a test compound are contacted with the galanin receptorprotein expressed on the cell membrane by culturing the transformantcontaining the galanin receptor protein-encoding DNA, the resultinggalanin receptor protein-mediated cell stimulating activities(activities of promoting or activities of inhibiting physiologicalresponses such as liberation of arachidonic acid, liberation ofacetylcholine, endocellular Ca²⁺ liberation, endocellular cAMPproduction, endocellular cGMP production, production of inositolphosphate, changes in cell membrane potential, phosphorylation ofendocellular proteins, activation of c-fos, lowering of pH, insulinsecretion, etc.) are measured and compared: are provided.

[0288] In the above-mentioned screening methods {circle over (1)} or{circle over (2)} (a), a compound which binds with a galanin receptorprotein (e.g., human galanin receptor protein, etc.) and inhibits thebinding of galanin with the galanin receptor protein can be selected asa galanin receptor agonist or antagonist.

[0289] Further, in the above-mentioned screening method {circle over(2)} (b), a compound which exhibits a cell-stimulating activity (forexample, activities of promoting or inhibiting the opening of K⁺channel, closing of N type Ca⁺ channel, liberation of arachidonic acid,liberation of acetylcholine, variations in intracellular Ca²⁺concentration, inhibition of intracellular cAMP production, productionof inositol phosphate, variations in cell membrane potential,phosphorylation of intracellular protein, activation of c-fos, decreasein pH, cell migration activity, secretion of hormones, activation of Gprotein, cell promulgation, etc.) via the galanin receptor (e.g., humangalanin receptor, etc.) upon said galanin receptor binding can beselected as a galanin receptor agonist.

[0290] On the other hand, in the above-mentioned screening methods{circle over (1)} and {circle over (2)} (a), a compound having nostimulating activity to said cells among the test compounds whichexhibit an activity of inhibiting the binding of galanin with thegalanin protein receptor (e.g., human galanin receptor protein, etc.)can be selected as a galanin receptor antagonist.

[0291] Before the cells containing the galanin receptor protein (e.g.,human galanin receptor protein, etc.) of the present invention weredeveloped, there was no cell which highly expressed the galanin receptorprotein (e.g., human galanin receptor protein, etc.) and, therefore, ithas not been possible to conduct an effective screening of galaninreceptor agonists or antagonists.

[0292] Specific explanations of the screening method will be given ashereunder.

[0293] First, with respect to the galanin receptor protein such as themouse-derived galanin receptor protein used for the screening method ofthe present invention, any product may be used so far as it containsgalanin receptor proteins or partial peptide thereof such asmouse-derived galanin receptor proteins or partial peptide thereofalthough the use of a membrane fraction derived from mammalian organs,tissues, cells, including mouse, is suitable. Galanin receptor proteinswhich are expressed in a large amount using a recombinant are suitablefor the screening.

[0294] In the manufacture of the galanin receptor protein (for example,mouse-derived galanin receptor protein, etc.), the above-mentionedmethod can be used and it may be carried out by expressing the DNAcoding for said protein in mammalian cells or in insect cells. Withrespect to the DNA fragment coding for the target region, complementaryDNA may be used although it is not limited thereto. Thus, for example,gene fragments or synthetic DNA may be used as well.

[0295] In order to introduce the galanin receptor protein-encoding DNAfragment (for example, mouse-derived galanin receptor protein-encodingDNA fragment, etc.) into host animal cells and to express itefficiently, it is preferred that said DNA fragment is incorporated intothe downstream of polyhedron promoter of nuclear polyhedrosis virusbelonging to baculovirus, promoter derived from SV40, promoter ofretrovirus, metallothionein promoter, human heat shock promoter,cytomegalovirus promoter, SRα promoter, etc. Examinations of thequantity and the quality of expressed receptors can be carried out byknown methods per se or modified methods substantially analogousthereto. For example, they may be conducted by the method described inpublications such as Nambi, P. et al.: The Journal of BiochemicalSociety, vol.267, pages 19555-19559 (1992).

[0296] Accordingly, in the screening method, the substance containing agalanin receptor protein or a partial peptide thereof (for example,mouse-derived galanin receptor protein or its partial peptide, etc.) maybe a galanin receptor protein or its partial peptide (for example,mouse-derived galanin receptor protein or its partial peptide, etc.)which is purified by known methods per se or a cell containing saidprotein or a cell membrane fraction of the cell containing said protein,etc.

[0297] When the galanin receptor protein-containing cells are used inthe screening method, said cells may be immobilized with glutaraldehyde,formalin, etc. The immobilization may be carried out by known methodsper se or modified methods substantially analogous thereto.

[0298] For example, the mouse-derived galanin receptorprotein-containing cells are host cells expressing the mouse-derivedgalanin receptor, naturally occurring cells containing the mouse-derivedgalanin receptor protein, etc. Examples of said host cells may includeEscherichia coli, Bacillus subtilis, yeasts, insect cells, animal cellssuch as CHO cell and COS cell, etc. The host cell expressing themouse-derived galanin receptor can be produced by the method accordingto the above-mentioned transformant production.

[0299] In conducting the above-mentioned methods {circle over (1)},{circle over (2)} (a) and {circle over (3)} for screening the galaninreceptor agonist and/or galanin receptor antagonist, a suitable galaninreceptor fraction and a labeled galanin are necessary. With respect tothe galanin receptor fraction, it is preferred to use naturallyoccurring galanin receptors (natural type galanin receptors),recombinant type galanin receptor fractions with the activity equivalentto that of the natural type galanin receptor, cells expressing therecombinant type mouse-derived galanin receptor, naturally occurringcells containing the mouse-derived galanin receptor, etc. Here the term“activity equivalent to” means the same galanin binding activity, or thesubstantially equivalent galanin binding activity.

[0300] With respect to the labeled galanin, it is possible to uselabeled galanin, labeled galanin analogized compounds, etc. For example,galanin labeled with [³H], [¹²⁵I], [¹⁴C], [³⁵S], etc. and other labeledsubstances may be utilized. Known galanin agonists and antagonistslabeled with [³H], [¹²⁵I], [¹⁴C], etc. may be utilized. Preferredexamples of the labeled galanin are galanin labeled with [¹²⁵I](Dupont/NEN), etc.

[0301] Specifically, galanin receptor protein-containing cells or cellmembrane fractions (for example, human or mouse galanin receptorprotein-containing cells or cell membrane fractions of the presentinvention) or the galanin receptor proteins or partial peptides thereofare first suspended in a buffer which is suitable for the measuringmethod to prepare the receptor sample in conducting the screening for agalanin receptor agonist and/or galanin receptor antagonist. Withrespect to the buffer, any buffer such as Tris-HCl buffer or phosphatebuffer of pH 4-10 (preferably, pH 6-8) which does not inhibit thebinding of galanin with the receptor may be used.

[0302] In addition, a surface-active agent such as CHAPS, Tween 80™(Kao-Atlas, Japan), digitonin, deoxycholate, etc. and/or variousproteins such as bovine serum albumin (BSA), gelatin, etc. may be addedto the buffer with an object of decreasing the nonspecific binding.Further, a protease inhibitor such as PMSF, leupeptin, bacitracin,aprotinin, E-64 (manufactured by Peptide Laboratory, Japan), pepstatin,etc. may be added with an object of inhibiting the decomposition of thereceptor and galanin by protease. A labeled galanin in a certain amount(for example, about 10,000 cpm to 1,000,000 cpm in case of 2000Ci/mmol;5,000 cpm to 500,000 cpm in other cases) is added to 0.01 ml to 10 ml ofsaid receptor solution and, at the same time, 10⁻⁴ M to 10⁻¹⁰ M of atest compound coexists. In order to determine the nonspecific bindingamount (NSB), a reaction tube to which a great excessive amount ofunlabeled test compounds is added is prepared as well.

[0303] The reaction is carried out at 0-50° C. (preferably at 4-37° C.)for 20 minutes to 24 hours (preferably 30 minutes to three hours). Afterthe reaction, it is filtered through a glass fiber filter, a filterpaper, or the like, washed with a suitable amount of the same buffer andthe radioactivity (for example, the amount of [¹²⁵I], etc.) retained inthe glass fiber filter, etc. is measured by means of a liquidscintillation counter or a γ-counter. Although a manifold or a cellharvester may be used for the filtration, the use of cell harvester isrecommended for improving the efficiency. Supposing that the count(B₀-NSB) obtained by subtracting the nonspecific binding amount (NSB)from the total binding amount (B₀) wherein an antagonizing substance isnot present is set at 100%, the test compound in which the specificbinding amount (B-NSB) obtained by subtracting the nonspecific bindingamount (NSB) from the total binding amount (B) is, for example, lessthan 50% may be selected as a inhibitory candidate substance, i.e.,agonist and/or antagonist candidate compound.

[0304] In an embodiment of the screening using human galanin receptorproteins, the operation is carried out in accordance with the followingprocedures:

[0305] (i) A reaction buffer (pH: 7.4) comprising 20 mM of Tris-HCl, 1mM of EDTA, 0.1% of BSA, 0.05% of CHAPS, 0.5mM of PMSF, 40 μg/ml ofleupeptin, 20 μg/ml of E-64 and 10 μg/ml of pepstatin is prepared.

[0306] (ii) A test compound solution (2 μl) in which the test compoundis suspended in the reaction buffer is placed in a reaction tube on anice bath. The final concentration of the test compound is adjusted to100 μM.

[0307] (iii) The cell membrane fraction containing human galaninreceptor protein freeze-dried at −80° C. is returned to a roomtemperature and then vortex is gently generated and diluted to asuitable concentration to prepare a cell membrane fraction solution (forexample, 0.5 mg protein/ml (bovine hippocampal membrane fraction, CHOcell membrane fraction) etc.). This cell membrane fraction solution ispassed through a cell strainer and each 200 μl of it is placed in eachreaction tube using a separator.

[0308] (iv) Each 2 μl of [¹²⁵I] galanin diluted in a reaction buffer isplaced in a reaction tube on an ice bath.

[0309] (v) The reaction is carried out at 25° C. for 60 or 75 minutes.

[0310] (vi) A B/F separation is conducted using a manifold. The filter(GF/F, Whatman) which is used therefor is previously dipped in a PEIsolution (20 mM Tris-HCl and 0.3% polyethyleneimide; pH: 7.4) for morethan one hour.

[0311] (vii) The filter is counted using a gamma-counter. The compoundwhich inhibits the specific binding to an extent of 40-50% or more andof 50% or more is evaluated as±and +, respectively.

[0312] In conducting the above-mentioned methods {circle over (2)} (b)and {circle over (4)} for screening the galanin receptor agonist and/orgalanin receptor antagonist, the galanin receptor protein (e.g.,mouse-derived galanin receptor protein)-mediated cell stimulatingactivity (e.g., activities of promoting or activities of inhibitingphysiological responses such as liberation of arachidonic acid,liberation of acetylcholine, endocellular Ca²⁺ liberation, endocellularcAMP production, endocellular cGMP production, production of inositolphosphate, changes in the cell membrane potential, phosphorylation ofendocellular proteins, activation of c-fos, lowering of pH, secretion ofinsulin, etc.) may be measured by known methods or by the use ofcommercially available measuring kits.

[0313] In conducting a screening method of the above-mentioned {circleover (2)} (b), it is possible to measure the cell stimulating activityvia the galanin receptor protein (e.g., human galanin receptor protein,etc.) (for example, activities of promoting or inhibiting the opening ofK⁺ channel, closing of N type Ca⁺ channel, liberation of arachidonicacid, liberation of acetylcholine, variations in intracellular Ca²⁺concentration, inhibition of intracellular cAMP production, productionof inositol phosphate, variations in cell membrane potential,phosphorylation of intracellular protein, activation of c-fos, decreasein pH, cell migration activity, secretion of hormones, activation of Gprotein, cell promulgation, etc.) by known method or bycommercially-available measuring kits. To be more specific, galaninreceptor protein (e.g., human or mouse-derived galanin receptor protein,etc.)-containing cells are at first cultured in a multiwell plate or thelike.

[0314] In conducting the screening, it is substituted with a suitablebuffer which does not show toxicity to fresh media or cells in advance,incubated for a certain period after adding a test compound, etc.thereto. The resultant cells are extracted or the supernatant liquid isrecovered and the resulting product is determined, preferablyquantitatively, by each of the methods. When it is difficult to identifythe production of the index substance (e.g. arachidonic acid, etc.)which is to be an index for the cell stimulating activity due to thepresence of decomposing enzymes contained in the cell, an assay may becarried out by adding an inhibitor against said decomposing enzyme. Withrespect to the activities such as an inhibitory action against cAMPproduction, it may be detected as an inhibitory action against the cAMPproduction in the cells whose fundamental production has been increasedby forskolin or the like.

[0315] In conducting a screening by measuring the cell stimulatingactivity, cells in which a suitable galanin receptor protein (e.g.,mouse-derived galanin receptor protein) is expressed are necessary.Preferred galanin receptor protein (e.g., mouse-derived galanin receptorprotein)-expressing cells are naturally occurring mouse-derived galaninreceptor protein (natural type mouse-derived galanin receptorprotein)-containing cell lines or strains (e.g., mouse MIN6 (FERMBP-4954), etc.), the above-mentioned recombinant type mouse-derivedgalanin receptor protein-expressing cell lines or strains, etc. Amongthem, the natural type mouse-derived galanin receptor protein-containingcell line, mouse pancreas-derived MIN6 cell, is capable of secretinginsulin from intracellular regions when galanin binds with galaninreceptors on the cell membrane of said cell. In case where the insulinsecretion is used as an index for the cell stimulating activity, mousepancreas-derived MIN6 cell is particularly preferred.

[0316] Examples of the test compound includes peptides, proteins,non-peptidic compounds, synthesized compounds, fermented products, cellextracts, plant extracts, animal tissue extracts, serum, blood, bodyfluid, etc. Those compounds may be novel or known.

[0317] A kit for screening the galanin receptor agonist and/or galaninreceptor antagonist comprises a galanin receptor protein or a saltthereof according to the present invention (e.g., human or mouse-derivedgalanin receptor protein or its salt) or a partial peptide thereofaccording to the present invention (e.g., human or mouse-derived galaninreceptor partial peptide or its salt), a galanin receptor protein (e.g.,human or mouse-derived galanin receptor protein)-containing cell or itscell membrane fraction according to the present invention, etc.

[0318] Examples of the screening kit include as follows:

[0319] 1. Reagent for Determining Ligand.

[0320] {circle over (1)} Buffer for Measurement and Buffer for Washing.

[0321] The product wherein 0.05% of bovine serum albumin (manufacturedby Sigma) is added to Hanks' Balanced Salt Solution (manufactured byGibco).

[0322] This may be sterilized by filtration through a membrane filterwith a 0.45 μm pore size, and stored at 4° C. or may be prepared uponuse.

[0323] {circle over (2)} Sample of Mouse-Derived Galanin ReceptorProtein.

[0324] CHO cells in which a mouse-derived galanin receptor protein isexpressed are subcultured at the rate of 5×10⁵ cells/well in a 12-wellplate and cultured at 37° C. with a 5% CO₂ and 95% air atmosphere fortwo days to prepare the sample.

[0325] {circle over (3)} Labeled Galanin.

[0326] The galanin which is labeled with commercially available [³H],[¹²⁵I], [¹⁴C], [³⁵S], etc.

[0327] The product in a state of an aqueous solution is stored at 4° C.or at −20° C. and, upon use, diluted to 5 μM with a buffer for themeasurement.

[0328] {circle over (4)} Standard Galanin Solution.

[0329] Galanin is dissolved in PBS containing 0.1% of bovine serumalbumin (manufactured by Sigma) to make 100 μM and stored at −20° C.

[0330] 2. Method of the Measurement.

[0331] {circle over (1)} CHO cells are cultured in a 12-well tissueculture plate to express mouse-derived galanin receptor proteins. Thereceptor protein-expressing CHO cells are washed with 1 ml of buffer forthe measurement twice. Then 490 μl of buffer for the measurement isadded to each well.

[0332] {circle over (2)} Five μl of a test compound solution of 10⁻³ to10⁻¹⁰ M is added, then 5 μl of a labeled galanin is added and is made toreact at room temperature for one hour. For knowing the non-specificbinding amount, 5 μl of the galanin of 10⁻⁴ M is added instead of thetest compound.

[0333] {circle over (3)} The reaction solution is removed from the well,which is washed with 1 ml of buffer for the measurement three times. Thelabeled ligand binding with the cells is dissolved in 0.2N NaOH-1% SDSand mixed with 4 ml of a liquid scintillator A (manufactured by WakoPure Chemical, Japan).

[0334] {circle over (4)} Radioactivity is measured using an automaticγ-counter or a liquid scintillation counter (manufactured by Beckmann)and PMB (percent of maximum binding) is calculated by the followingexpression (1):

PMB=[(B−NSB)/(B ₀ −NSB)]×100  (1)

[0335] PMB: Percent of maximum binding

[0336] B: Value when a sample is added

[0337] NSB: Nonspecific binding

[0338] B₀: Maximum binding

[0339] Another example of the screening kit is as follows:

Reagents for the Screening

[0340] {circle over (1)} Buffer for Measurement and for Washing.

[0341] Hanks buffer to which 0.01% of bovine serum albumin and 0.05% ofCHPS are added is used. This is filtered through a filter with a poresize of 0.22 μm, sterilized and stored at 4° C. or may be prepared uponuse.

[0342] {circle over (2)} Human Galanin Receptor Protein Sample.

[0343] Cells containing the human galanin receptor protein aresubcultured in a 12-well plate at 5×10⁵/well and cultured at 37° C. with5% CO₂ and 95% air until cells become confluent.

[0344] {circle over (3)} Labeled Galanin.

[0345] Commercially-available galanin which is labeled with [³H],[¹²⁵I], [¹⁴C], etc. is used. That which is in a state of solution isstored at 4° C. or −20° C. and, upon use, it is diluted to 1 μl with abuffer for the measurement.

[0346] {circle over (4)} Standard Galanin Solution.

[0347] Galanin is diluted with sterilized water to make 10⁻⁴ M andstored at −20° C.

Method of Measurement

[0348] {circle over (1)} Cells containing human galanin receptor proteincultured in a 12-well culturing plate are washed, about twice, with 1 mlof a buffer for measurement.

[0349] {circle over (2)} After the buffer for measurement is sucked out,5 μl of a test compound solution (10⁻³ to 10⁻¹⁰ M) cooled at 4° C. isadded, then 0.5 ml of a buffer for measurement, containing 100 pM of alabeled galanin, is added and the mixture is made to react at 37° C. forone hour with 5% CO₂ and 95% air. In order to know the nonspecificbinding amount, 1 μM of galanin is added together with the testcompound.

[0350] {circle over (3)} The reaction solution is removed and washed,three times, with 1 ml of buffer for washing which is kept at 37° C. Thelabeled galanin bonded to the cells is removed with 0.5 ml of 0.2N NaOHand the radioactivity is measured by a γ-counter to calculate the PMB(percent of maximum binding) from the above formula (1).

[0351] In the above-mentioned screening methods and screening kit, it isalso possible to use recombinant human galanin receptor proteinmanufactured from DNA such as known human galanin receptor protein DNAor the like or partial peptide thereof or cells containing saidrecombinant human galanin receptor protein or a cell membrane fractionthereof instead of the human galanin receptor protein of the presentinvention or the partial peptide thereof or cells containing the humangalanin receptor protein or a cell membrane fraction thereof.

[0352] The compound or its salt obtained by the screening method orscreening kit of the present invention is a compound which inhibits thebinding of galanin with a galanin receptor protein and, moreparticularly, it is a compound having a cell stimulating activitymediated via a galanin receptor or a salt thereof (so-called “galaninreceptor agonist”) or a compound having no said stimulating activity(so-called “galanin receptor antagonist”). Examples of said compound arepeptides, proteins, non-peptidic compounds, synthesized compounds,fermented products, etc. and the compound may be novel or known. Thegalanin receptor agonist or antagonist obtained by the screening methodor the screening kit of the present invention is a compound or saltthereof selected from the test sample including the compounds (forexample, peptides, proteins, nonpeptidic compounds, synthesizedcompounds, fermented products, cell extracts, plant extracts, animaltissue extracts, cell or tissue cultures, biological fluids, etc.; saidtest compounds may be either novel or known) using the screening methodor the screening kit of the present invention and is a compound whichinhibits the binding of galanin with the recombinant human galaninreceptor protein of the present invention. Among those compounds,galanin receptor agonist is a compound which exhibits a cell-stimulatingactivity via a human galanin receptor while galanin receptor antagonistis a compound which does not exhibit said cell-stimulating activity.

[0353] In addition, the compounds in which the structure of said galaninreceptor agonist or antagonist obtained by the screening method or thescreening kit of the present invention is chemically modified orsubstituted or the compounds in which a design is conducted based uponsaid structure are also included in the galanin receptor agonist orantagonist obtained by the screening method or the screening kit of thepresent invention.

[0354] With respect to the salt of said galanin receptor agonist orantagonist, physiologically-acceptable acid addition salts thereof areparticularly preferred. Examples of such salts are those with inorganicacids (for example, hydrochloric acid, phosphoric acid, hydrobromic acidand sulfuric acid) or with organic acids (for example, acetic acid,formic acid, propionic acid, fumaric acid, maleic acid, succinic acid,tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid and benzenesulfonic acid).

[0355] The galanin receptor agonist exhibits all or part of thephysiological activities of galanin or its equivalent and, therefore, itis useful as an active ingredient for a pharmaceutical composition withsafety and low toxicity depending upon said physiological activities. Onthe other hand, the galanin receptor antagonist may inhibit all or partof the physiological activities of galanin or its equivalent and,therefore, it is useful as an active ingredient for a pharmaceuticalcomposition with safety and low toxicity inhibiting said physiologicalactivities.

[0356] More specifically, galanin receptor agonists are useful as aninhibitor for liberation of acetylcholine, an inhibitor for insulinsecretion, a stimulant for growth hormone secretion, an inhibitor forlearning behavior or an inhibitor for satiety, etc. and, moreover, it isuseful as a preventive and therapeutic agent for schizophrenic disease,gastric ulcer, as a sedative, etc. On the other hand, galanin receptorantagonists are useful as an accelerator for liberation ofacetylcholine, an accelerator for insulin secretion, an inhibitor forgrowth hormone secretion, an accelerator for learning behavior and anaccelerator for satiety, etc. and, moreover, it is useful as apreventive and therapeutic agent for diabetes, Alzheimer's disease,dementia, etc.

[0357] When the galanin receptor agonist and/or galanin receptorantagonist or the salt thereof obtained by the screening method or bythe screening kit is used as the above-mentioned pharmaceuticalcomposition, a conventional means may be applied therefor. The compoundor the salt thereof may be orally, parenterally, by inhalation spray,rectally, or topically administered as pharmaceutical compositions orformulations (e.g. powders, granules, tablets, pills, capsules,injections, syrups, emulsions, elixirs, suspensions, solutions, etc.).For example, it may be used by an oral route as tablets (sugar-coated ifnecessary), capsules, elixirs, microcapsules, etc. or by a parenteralroute as injections such as an aseptic solution or a suspension in wateror in other pharmaceutically acceptable liquid. The pharmaceuticalcompositions or formulations may comprise at least one such compoundalone or in admixture with pharmaceutically acceptable carriers,adjuvants, vehicles, excipients and/or diluents. The pharmaceuticalcompositions can be formulated in accordance with conventional methods.For example, said compound or the salt thereof is mixed in a unit doseform which is required for preparing a generally approved pharmaceuticalpreparations together with a physiologically acceptable carriers,flavoring and/or perfuming agents (fragrances), fillers, vehicles,antiseptics, stabilizers, binders, etc. whereupon the preparation can bemanufactured. An amount of the effective component in those preparationsis to be in such an extent that the suitable dose within an indicatedrange is achieved.

[0358] Examples of the additives which can be admixed in the tablets,capsules, etc. are binders such as gelatin, corn starch, tragacanth andgum arabicum; fillers such as crystalline cellulose; swelling agentssuch as corn starch, gelatin and alginic acid; lubricants such asmagnesium stearate; sweetening agents such as sucrose, lactose andsaccharine; preservatives such as parabens and sorbic acid; antioxidantssuch as ascorbic acid, α-tocopherol and cysteine; fragrances such aspeppermint, akamono oil and cherry; disintegrants; buffering agents;etc. Other additives may include mannitol, maltitol, dextran, agar,chitin, chitosan, pectin, collagen, casein, albumin, synthetic orsemi-synthetic polymers, glyceride, lactide, etc. When the unit form ofthe preparation is a capsule, a liquid carrier such as fat/oil may befurther added besides the above-mentioned types of materials. Theaseptic composition for injection may be formulated by a conventionaltechnique or practice for the preparations such as that the activesubstance in a vehicle such as water for injection is dissolved orsuspended in a naturally occurring plant oil such as sesame oil and palmoil.

[0359] Examples of an aqueous liquid for the injection are aphysiological saline solution and isotonic solutions containing glucoseand other auxiliary agents (e.g. D-sorbitol, D-mannitol, sodiumchloride, etc.) wherein a suitable auxiliary solubilizers such asalcohol (e.g. ethanol, etc.), polyalcohol (e.g. propylene glycol,polyethylene glycol, etc.), nonionic surface-active agent (e.g.Polysorbate 80™, HCO-50, etc.), etc. may be jointly used. In the case ofthe oily liquid, sesame oil, soybean oil, etc. may be exemplifiedwherein benzyl benzoate, benzyl alcohol, etc. may be jointly used asauxiliary solubilizers.

[0360] In addition, buffers (e.g. phosphate buffer, sodium acetatebuffer, etc.), analgesic agents (e.g. benzalkonium chloride, procainehydrochloride, etc.), stabilizers (e.g. human serum albumin,polyethylene glycol, etc.), stabilizers (e.g. benzyl alcohol, phenol,etc.), antioxidants, etc. may be compounded therewith too. The preparedinjection solution is filled in suitable ampoules. The formulationprepared as such is safe and less toxic and, therefore, it can beadministered to warm-blooded mammals such as rats, rabbits, sheep,swines, cattle, cats, dogs, monkeys, human being, etc.

[0361] Dose levels of said galanin receptor agonist and/or galaninreceptor antagonist or the salt thereof may vary depending upon thesymptom. Specific dose levels for any particular patient will beemployed depending upon a variety of factors including the activity ofspecific compounds employed, the age, body weight, general health, sex,diet, time of administration, route of administration, rate ofexcretion, drug combination, and the severity of the particular diseaseundergoing therapy. In the case of oral administration, it is usuallyabout 0.1-100 mg, preferably about 1.0-50 mg or, more preferably, about1.0-20 mg per day for adults (as 60 kg). When it is administeredparenterally, its dose at a time may vary depending upon the object tobe administered, organs to be administered, symptoms, administeringmethods, etc. The term “parenteral” as used herein includes subcutaneousinjections, intravenous, intramuscular, intraperitoneal injections, orinfusion techniques. In the case of injections, it is usually convenientto give by an intravenous route in an amount of about 0.01-30 mg,preferably about 0.1-20 mg or, more preferably, about 0.1-10 mg per dayto adults (as 60 kg). In the case of other animals, the dose calculatedfor 60 kg may be administered as well.

[0362] (3) Preventive and Therapeutic Agent for of Galanin ReceptorProtein Deficiency Diseases

[0363] The galanin receptor protein (e.g., human galanin receptorprotein)-encoding DNA can be used a preventive and/or therapeutic agentfor treating said galanin receptor protein deficiency diseases.

[0364] For example, when there is a patient for whom the physiologicalaction of galanin cannot be expected because of a decrease in thegalanin receptor protein (e.g., human galanin receptor protein) in vivo,the amount of the galanin receptor protein in the cells of said patientcan be increased whereby the action of galanin can be fully achieved by:

[0365] (a) administering the galanin receptor protein (e.g., humangalanin receptor protein)-encoding DNA to the patient to express it; or

[0366] (b) inserting the galanin receptor protein (e.g., human galaninreceptor protein)-encoding DNA into cells or the like to express it,followed by transplanting said cells or the like to said patient.Accordingly, the galanin receptor protein (e.g., human galanin receptorprotein)-encoding DNA can be used as a safe and less toxic preventiveand therapeutic agent for the galanin receptor protein (e.g., humangalanin receptor protein) deficiency diseases (e.g., diabetes,Alzheimer's disease, dementia, etc.). It may be used in treating orremedying defects by promoting the acetylcholine liberation, inhibitingthe growth hormone secretion, promoting the insulin secretion, promotingthe learning behavior, promoting satiety, etc.

[0367] When the DNA of the present invention is used as theabove-mentioned agent, said DNA may be used alone or after inserting itinto a suitable vector such as retrovirus vector, adenovirus vector,adenovirus-associated virus vector, etc. followed by subjecting theproduct vector to a conventional means. Thus, it may be administeredorally parenterally, by inhalation spray, rectally, or topically aspharmaceutical compositions or formulations. Oral formulations includetablets (sugar-coated if necessary), capsules, elixirs, microcapsules,etc. Parenteral formulations include injections such as an asepticsolution or a suspension in water or in other pharmaceuticallyacceptable liquid. For example, the DNA of the present invention isadmixed in a unit dose form which is required for preparing generallyapproved pharmaceutical preparations together with a physiologicallyacceptable carriers, flavoring agents, adjuvants, excipients, diluents,fillers, vehicles, antiseptics, stabilizers, binders, etc. whereupon thepreparation can be manufactured. The amount of the effective componentin those preparations is to be in such an extent that the suitable dosewithin an indicated range is achieved.

[0368] Examples of the additives which can be admixed in the tablets,capsules, etc. are binders such as gelatin, corn starch, tragacanth andgum arabicum; fillers such as crystalline cellulose; swelling agentssuch as corn starch, gelatin and alginic acid; lubricating agents suchas magnesium stearate; sweetening agents such as sucrose, lactose andsaccharine; and flavoring agents such as pepper mint, akamono oil andcherry. When the unit dose form of the preparation is a capsule, aliquid carrier such as fat/oil may be further added in addition of theabove-mentioned types of materials. The aseptic composition forinjection may be formulated by conventional practices for thepreparations such as that the active substance in a vehicle such aswater for injection is dissolved or suspended in naturally occurringplant oil such as sesame oil and palm oil.

[0369] Examples of an aqueous liquid for injection are a physiologicalsaline solution and isotonic solutions containing glucose and otherauxiliary agents (e.g. D-sorbitol, D-mannitol, sodium chloride, etc.)wherein a suitable auxiliary solubilizers such as alcohol (e.g. ethanol,etc.), polyalcohol (e.g. propylene glycol polyethylene glycol, etc.),nonionic surface-active agent (e.g. Polysorbate 80™, HCO-50, etc.), etc.may be jointly used. Examples of an oily liquid include sesame oil,soybean oil, etc. wherein benzyl benzoate, benzyl alcohol, etc. may bejointly used as auxiliary solubilizers. In addition, buffers (e.g.phosphate buffer, sodium acetate buffer, etc.), analgesic agents (e.g.benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g.human serum albumin, polyethylene glycol, etc.), stabilizers (e.g.benzyl alcohol phenol, etc.), antioxidants, etc. may be admixedtherewith too. The prepared injection solution is filled in suitableampoules. The preparation prepared as such is safe and less toxic and,therefore, it can be administered to warm-blooded animals (e.g., rat,rabbit, sheep, swine, cattle, cat, dog, monkey, human beings, etc.).

[0370] Specific dose levels of said DNA may vary depending upon avariety of factors including the activity of drugs employed, the age,body weight, general health, sex, diet, time of administration, route ofadministration, drug combination, and the severity of the symptom. Inthe case of oral administration, it is usually about 0.1-100 mg,preferably about 1.0-50 mg or, more preferably, about 1.0-20 mg per dayfor adults (as 60 kg). When it is administered parenterally, its dose ata time may vary depending upon the object (patient) to be administered,organs to be administered, symptoms, administering methods, etc. but, inthe case of injections, it is usually convenient to give by anintravenous route in an amount of about 0.01-30 mg, preferably about0.1-20 mg or, more preferably, about 0.1-10 mg per day to adults (as 60kg). For other animals, the dose calculated from the above based uponthe body weight may be administered.

[0371] Furthermore, the method of inserting the DNA of the presentinvention into cells to express said galanin receptor protein (e.g.,human galanin receptor protein, etc.) followed by transplanting saidcells to the patient may be carried out by a method which is known perse or is similar thereto.

[0372] (4) Manufacture of Antibody or Antiserum against the GalaninReceptor Protein of the Present Invention, Its Partial Peptide or ItsSalt.

[0373] Antibodies (e.g. polyclonal antibody and monoclonal antibody) andantisera against the galanin receptor protein or salt thereof of thepresent invention or against the peptide fragment of the galaninreceptor protein or salt thereof of the present invention may bemanufactured by antibody or antiserum-manufacturing methods per se knownto those of skill in the art or methods similar thereto, using thegalanin receptor protein or its salt of the present invention or thepartial peptide (fragment) of the galanin receptor protein or its saltof the present invention. For example, monoclonal antibodies can bemanufactured by the method as given herein below.

Preparation of Monoclonal Antibody

[0374] (a) Preparation of Monoclonal Antibody-Producing Cells.

[0375] The galanin receptor protein of the present invention or its saltor the partial peptide of the galanin receptor protein of the presentinvention or its salt is administered to warm-blooded animals eithersolely or together with carriers or diluents to the site where theproduction of antibody is possible by the administration. In order topotentiate the antibody productivity upon the administration, completeFreund's adjuvants or incomplete Freund's adjuvants may be administered.The administration is usually carried out once every two to six weeksand two to ten times in total. Examples of the applicable warm-bloodedanimals are monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep,goats chickens and hamsters and the use of mice, rats and hamsters ispreferred.

[0376] In the preparation of the cells which produce monoclonalantibodies, an animal wherein the antibody titer is noted is selectedfrom warm-blooded animals (e.g. mice) immunized with antigens, thenspleen or lymph node is collected after two to five days from the finalimmunization and antibody-producing cells contained therein are fusedwith myeloma cells to give monoclonal antibody-producing hybridomas.Measurement of the antibody titer in antisera may, for example, becarried out by reacting a labeled galanin receptor protein (which willbe mentioned later) with the antiserum followed by measuring the bindingactivity of the labeling agent with the antibody. The operation forfusing may be carried out, for example, by a method of Koehler andMilstein (Nature, 256, 495, 1975). Examples of the fusion acceleratorare polyethylene glycol (PEG), Sendai virus, etc. and the use of PEG ispreferred.

[0377] Examples of the myeloma cells are NS-1, P3U1, SP2/0, AP-1, etc.and the use of P3U1 is preferred. The preferred fusion ratio of thenumbers of antibody-producing cells used (spleen cells) to the numbersof myeloma cells is within a range of about 1:1 to 20:1. When PEG(preferably, PEG 1000 to PEG 6000) is added in a concentration of about10-80% followed by incubating at 20-40° C. (preferably, at 30-37° C.)for one to ten minutes, an efficient cell fusion can be carried out.

[0378] Various methods may be applied for screening a hybridoma whichproduces anti-galanin receptor antibody. For example, a supernatantliquid of hybridoma culture is added to a solid phase (e.g. microplate)to which the galanin receptor protein antigen is adsorbed eitherdirectly or with a carrier, then anti-immunoglobulin antibody(anti-mouse immunoglobulin antibody is used when the cells used for thecell fusion are those of mouse) which is labeled with a radioactivesubstance, an enzyme or the like, or protein A is added thereto and thenanti-galanin receptor monoclonal antibodies bound on the solid phase aredetected; or a supernatant liquid of the hybridoma culture is added tothe solid phase to which anti-immunoglobulin or protein A is adsorbed,then the galanin receptor labeled with a radioactive substance or anenzyme is added and anti-galanin receptor monoclonal antibodies bondedwith the solid phase is detected.

[0379] Selection and cloning of the anti-galanin receptor monoclonalantibody-producing hybridoma may be carried out by methods per se knownto those of skill in the art or methods similar thereto. Usually, it iscarried out in a medium for animal cells, containing HAT (hypoxanthine,aminopterin and thymidine). With respect to a medium for the selection,for the cloning and for the growth, any medium may be used so far ashybridoma is able to grow therein. Examples of the medium are an RPMI1640 medium (Dainippon Pharmaceutical Co., Ltd., Japan) containing 1-20%(preferably 10-20%) of fetal calf serum (FCS), a GIT medium (Wako PureChemical, Japan) containing 1-20% of fetal calf serum and a serum-freemedium for hybridoma culturing (SFM-101; Nissui Seiyaku, Japan). Theculturing temperature is usually 20-40° C. and, preferably, about 37° C.The culturing time is usually from five days to three weeks and,preferably, one to two weeks. The culturing is usually carried out in 5%carbon dioxide gas. The antibody titer of the supernatant liquid of thehybridoma culture may be measured by the same manner as in theabove-mentioned measurement of the antibody titer of the anti-galaninreceptor in the antiserum.

[0380] The cloning can be usually carried out by methods known per sesuch as techniques in semi-solid agar and limiting dilution. The clonedhybridoma is preferably cultured in modern serum-free culture media toobtain optimal amounts of antibody in supernatants. The targetmonoclonal antibody is also preferably obtained from ascitic fluidderived from a mouse, etc. injected intraperitoneally with livehybridoma cells.

[0381] (b) Purification of the Monoclonal Antibody.

[0382] Like in the separation/purification of conventional polyclonalantibodies, the separation/purification of the anti-galanin receptormonoclonal antibody may be carried out by methods forseparating/purifying immunoglobulin (such as salting-out, precipitationwith an alcohol, isoelectric precipitation, electrophoresis, adsorption/deadsorption using ion exchangers such as DEAE, ultracentrifugation, gelfiltration, specific purifying methods in which only an antibody iscollected by treatment with an active adsorbent (such as anantigen-binding solid phase, protein A or protein G) and the bond isdissociated whereupon the antibody is obtained.

[0383] The galanin receptor antibody of the present invention which ismanufactured by the aforementioned method (a) or (b) is capable ofspecifically recognizing galanin receptors and, accordingly, it can beused for a quantitative determination of the galanin receptor in testliquid samples and particularly for a quantitative determination bysandwich immunoassays.

[0384] Thus, the present invention provides, for example, the followingmethods:

[0385] (i) a quantitative determination of a galanin receptor in a testliquid sample, which comprises

[0386] (a) competitively reacting the test liquid sample and a labeledgalanin receptor with an antibody which reacts with the galanin receptorof the present invention, and

[0387] (b) measuring the ratio of the labeled galanin receptor bindingwith said antibody; and

[0388] (ii) a quantitative determination of a galanin receptor in a testliquid sample, which comprises

[0389] (a) reacting the test liquid sample with an antibody immobilizedon an insoluble carrier and a labeled antibody simultaneously orcontinuously, and

[0390] (b) measuring the activity of the labeling agent on the insolublecarrier wherein one antibody is capable of recognizing the N-terminalregion of the galanin receptor while another antibody is capable ofrecognizing the C-terminal region of the galanin receptor.

[0391] When the monoclonal antibody of the present invention recognizinga galanin receptor (hereinafter, may be referred to as “anti-galaninreceptor antibody”) is used, galanin receptors can be measured and,moreover, can be detected by means of a tissue staining, etc. as well.For such an object, antibody molecules per se may be used or F(ab′)₂,Fab′ or Fab fractions of the antibody molecule may be used too. There isno particular limitation for the measuring method using the antibody ofthe present invention and any measuring method may be used so far as itrelates to a method in which the amount of antibody, antigen orantibody-antigen complex, depending on or corresponding to the amount ofantigen (e.g. the amount of galanin receptor, etc.) in the liquid sampleto be measured, is detected by a chemical or a physical means and thencalculated using a standard curve prepared by a standard solutioncontaining the known amount of antigen. For example, nephrometry,competitive method, immunometric method and sandwich method are suitablyused and, in terms of sensitivity and specificity, the sandwich methodwhich will be described herein later is particularly preferred.

[0392] Examples of the labeling agent used in the measuring method usingthe labeling substance are radioisotopes, enzymes, fluorescentsubstances, luminescent substances, colloids, magnetic substances, etc.Examples of the radioisotope are [¹²⁵I], [¹³¹I], [³H] and [¹⁴C];preferred examples of the enzyme are those which are stable and with bigspecific activity, such as β-galactosidase, β-glucosidase, alkaliphosphatase, peroxidase and malate dehydrogenase; examples of thefluorescent substance are fluorescamine, fluorescein isothiocyanate,etc.; and examples of the luminescent substance are luminol, luminolderivatives, luciferin, lucigenin, etc. Further, a biotin-avidin systemmay also be used for binding an antibody or antigen with a labelingagent.

[0393] In an insolubilization (immobilization) of antigens orantibodies, a physical adsorption may be used or a chemical bindingwhich is usually used for insolubilization or immobilization of proteinsor enzymes may be used as well. Examples of the carrier are insolublepolysaccharides such as agarose, dextran and cellulose; synthetic resinssuch as polystyrene, polyacrylamide and silicone; glass; etc.

[0394] In a sandwich (or two-site) method, the test liquid is made toreact with an insolubilized anti-galanin receptor antibody (the firstreaction), then it is made to react with a labeled anti-galanin receptorantibody (the second reaction) and the activity of the labeling agent onthe insoluble carrier is measured whereupon the amount of the galaninreceptor in the test liquid can be determined. The first reaction andthe second reaction may be conducted reversely or simultaneously or theymay be conducted with an interval. The type of the labeling agent andthe method of insolubilization (immobilization) may be the same as thosementioned already herein. In the immunoassay by means of a sandwichmethod, it is not always necessary that the antibody used for thelabeled antibody and the antibody for the solid phase is one type or onespecies but, with an object of improving the measuring sensitivity,etc., a mixture of two or more antibodies may be used too.

[0395] In the method of measuring galanin receptors by the sandwichmethod of the present invention, the preferred anti-galanin receptorantibodies used for the first and the second reactions are antibodieswherein their sites binding to the galanin receptors are different eachother. Thus, the antibodies used in the first and the second reactionsare those wherein, when the antibody used in the second reactionrecognizes the C-terminal region of the galanin receptor, then theantibody recognizing the site other than C-terminal regions, e.g.recognizing the N-terminal region, is preferably used in the firstreaction.

[0396] The anti-galanin receptor antibody of the present invention maybe used in a measuring system other than the sandwich method such as acompetitive method, an immunometric method and a nephrometry. In acompetitive method, an antigen in the test solution and a labeledantigen are made to react with an antibody in a competitive manner, thenan unreacted labeled antigen (F) and a labeled antigen binding with anantibody (B) are separated (i.e. B/F separation) and the labeled amountof any of B and F is measured whereupon the amount of the antigen in thetest solution is determined. With respect to a method for such areaction, there are a liquid phase method in which a soluble antibody isused as the antibody and the B/F separation is conducted by polyethyleneglycol, a second antibody to the above-mentioned antibody, etc.; and asolid phase method in which an immobilized antibody is used as the firstantibody or a soluble antibody is used as the first antibody while animmobilized antibody is used as the second antibody.

[0397] In an immunometric method, an antigen in the test solution and animmobilized antigen are subjected to a competitive reaction with acertain amount of a labeled antibody followed by separating into solidand liquid phases; or the antigen in the test solution and an excessamount of labeled antibody are made to react, then a immobilized antigenis added to bind an unreacted labeled antibody with the solid phase andseparated into solid and liquid phases. After that, the labeled amountof any of the phases is measured to determine the antigen amount in thetest solution.

[0398] In a nephrometry, the amount of insoluble sediment which isproduced as a result of the antigen-antibody reaction in a gel or in asolution is measured. Even when the antigen amount in the test solutionis small and only a small amount of the sediment is obtained, a lasernephrometry wherein scattering of laser is utilized can be suitablyused.

[0399] In applying each of those immunological measuring methods(immunoassays) to the measuring method of the present invention, it isnot necessary to set up any special condition, operation, etc. therefor.A measuring system (assay system) for galanin receptor may beconstructed taking the technical consideration of the persons skilled inthe art into consideration in the conventional conditions and operationsfor each of the methods. With details of those conventional technicalmeans, a variety of reviews, reference books, etc. may be referred to.They are, for example, Hiroshi Irie (ed): “Radioimmunoassay” (Kodansha,Japan, 1974); Hiroshi Irie (ed): “Radioimmunoassay; Second Series”(Kodansha, Japan, 1979); Eiji Ishikwa et al. (ed): “Enzyme Immunoassay”(Igaku Shoin, Japan, 1978); Eiji Ishikawa et al. (ed): “EnzymeImmunoassay” (Second Edition) (Igaku Shoin, Japan, 1982); Eiji Ishikawaet al. (ed): “Enzyme Immunoassay” (Third Edition) (Igaku Shoin, Japan,1987); “Methods in Enzymology” Vol. 70 (Immunochemical Techniques (PartA)); ibid. Vol. 73 (Immunochemical Techniques (Part B)); ibid. Vol. 74(Immunochemical Techniques (Part C)); ibid. Vol. 84 (ImmunochemicalTechniques (Part D: Selected Immunoassays)); ibid. Vol. 92(Immunochemical Techniques (Part E: Monoclonal Antibodies and GeneralImmunoassay Methods)); ibid. Vol. 121 (Immunochemical Techniques (PartI: Hybridoma Technology and Monoclonal Antibodies)) (Academic Press);etc.

[0400] As such, the amount of galanin receptor proteins can now bedetermined with a high precision using the anti-galanin receptorantibody of the present invention.

[0401] (5) Preparation of Animals Having the Galanin ReceptorProtein-Encoding DNA of the Present Invention.

[0402] It is possible to prepare transgenic animals expressing galaninreceptors using galanin receptor protein-encoding DNA. Examples of theanimals are warm-blooded mammals such as rats, mice, rabbit, sheep,swines, cattle, cats, dogs and monkeys.

[0403] In transferring the galanin receptor protein-encoding DNA to theaimed animal, it is generally advantageous that said DAN is used byligating with a site at the downstream of a promoter which is capable ofexpressing in animal cells. For example, when galanin receptor proteinDNA is to be transferred to a rabbit, a gene construct ligated with asite at the downstream of various promoters which are capable ofexpressing the galanin receptor protein DNA derived from an animalcompatible to the animal in animal host cells is subjected to amicroinjection to the fertilized ovum (oosperm) of the aimed animal(e.g. fertilized ovum (embryo) of rabbit) whereupon the transgenicanimal which produces the galanin receptor protein in a high amount canbe prepared.

[0404] Examples of the promoters used are promoters derived from virusand ubiquitous expression promoters such as metallothionein promotersmay be used but, preferably, enolase gene promoters and NGF genepromoters capable of specifically expressing in brain are used.

[0405] Transfer of the galanin receptor protein DNA at a fertilized ovumcell stage is secured in order that the DNA can be present in all ofembryonal cells and body somatic cells of an aimed animal. The fact thatthe galanin receptor protein is present in the fertilized ovum cells ofthe produced transgenic animal after the DNA transfer means that allprogeny of the produced transgenic animal have the galanin receptorprotein in all of their embryonal cells and somatic cells. Descendants(offsprings) of the animal of this type which inherited the gene havethe galanin receptor protein in all of their embryonal cells and somaticcells.

[0406] The transgenic animal to which the galanin receptor protein DNAis transferred can be subjected to a mating and a breeding forgenerations under a common breeding circumstance as the animal holdingsaid DNA after confirming that the gene can be stably retained.Moreover, male and female animals having the desired DNA are mated togive a homozygote having the transduced gene in both homologouschromosomes and then those male and female animals are mated whereby itis possible to breed for generations so that all descendants have saidDNA.

[0407] The animal to which the galanin receptor protein DNA istransferred highly expresses the galanin receptor protein and,accordingly, it is useful as the animal for screening for an agonist oran antagonist to said galanin receptor protein.

[0408] The DNA-transferred animal can be used as a cell source for atissue culture. For example, DNA or RNA in the tissue of theDNA-transferred mouse is directly analyzed or protein tissues expressedby gene are analyzed whereupon the galanin receptor protein can beanalyzed. Cells of the galanin receptor protein-containing tissue arecultured by standard tissue culture techniques whereupon it is possibleto study the function of the cells which are usually difficult toculture (e.g. those derived from brain and peripheral tissues) using theresulting culture. By using said cells, it is also possible to selectthe pharmaceuticals which can potentiate, for example, the functions ofvarious tissues. Moreover, if a cell strain with a high expression isavailable, it is possible to separate and purify galanin receptorproteins therefrom.

[0409] In the specification and drawings of the present application, theabbreviations used for bases (nucleotides), amino acids and so forth arethose recommended by the IUPAC-IUB Commission on BiochemicalNomenclature or those conventionally used in the art. Examples thereofare given below. Amino acids for which optical isomerism is possibleare, unless otherwise specified, in the L form.

[0410] DNA: Deoxyribonucleic acid

[0411] cDNA: Complementary deoxyribonucleic acid

[0412] A: Adenine

[0413] T: Thymine

[0414] G: Guanine

[0415] C: Cytosine

[0416] RNA: Ribonucleic acid

[0417] mRNA: Messenger ribonucleic acid

[0418] dATP: Deoxyadenosine triphosphate

[0419] dTTP: Deoxythymidine triphosphate

[0420] dGTP: Deoxyguanosine triphosphate

[0421] dCTP: Deoxycytidine triphosphate

[0422] ATP: Adenosine triphosphate

[0423] EDTA: Ethylenediamine tetraacetic acid

[0424] SDS: Sodium dodecyl sulfate

[0425] EIA: Enzyme Immunoassay

[0426] G, Gly: Glycine (or Glycyl)

[0427] A, Ala: Alanine (or Alanyl)

[0428] V, Val: Valine (or Valyl)

[0429] L, Leu: Leucine (or Leucyl)

[0430] I, Ile: Isoleucine (or Isoleucyl)

[0431] S, Ser: Serine (or Seryl)

[0432] T, Thr: Threonine (or Threonyl)

[0433] C, Cys: Cysteine (or Cysteinyl)

[0434] M, Met: Methionine (or Methionyl)

[0435] E, Glu: Glutamic acid (or Glutamyl)

[0436] D, Asp: Aspartic acid (or Aspartyl)

[0437] K, Lys: Lysine (or Lysyl)

[0438] R, Arg: Arginine (or Arginyl)

[0439] H, His: Histidine (or Histidyl)

[0440] F, Phe: Pheylalanine (or Pheylalanyl)

[0441] Y, Tyr: Tyrossine (or Tyrosyl)

[0442] W, Trp: Tryptophan (or Tryptophanyl)

[0443] P, Pro: Proline (or Prolyl)

[0444] N, Asn: Asparagine (or Asparaginyl)

[0445] Q, Gln: Glutamine (or Glutaminyl)

[0446] NVal: Norvaline (or Norvalyl)

[0447] pGlu: Pyroglutamic acid (or Pyroglutamyl)

[0448] Blc: γ-Butyrolacton-γ-carbonyl

[0449] Kpc: 2-Ketopiperidinyl-6-carbonyl

[0450] Otc: 3-Oxoperhydro-1,4-thiazin-5-carbonyl

[0451] Me: Methyl

[0452] Et: Ethyl

[0453] Bu: Butyl

[0454] Ph: Phenyl

[0455] TC: Thiazolidinyl-4(R)-carboxamide

[0456] The transformant Escherichia coli, designated JM109/p3H2-34,which is obtained in the Example 3 mentioned herein below, is on depositunder the terms of the Budapest Treaty from Oct. 12, 1994, with theNational Institute of Bioscience and Human-Technology (NIBH), Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry, Japan and has been assigned the Accession Number FERM BP-4828.It is also on deposit from Oct. 12, 1994 with the Institute forFermentation, Osaka, Japan (IFO) and has been assigned the AccessionNumber IFO 15749.

[0457] The transformant Escherichia coli, designated JM109/pMGR20, whichis obtained in the Example 4 mentioned herein below, is on deposit underthe terms of the Budapest Treaty from Dec. 15, 1994, with NIBH and hasbeen assigned the Accession Number FERM BP-4937. It is also on depositfrom Dec. 14, 1994 with IFO and has been assigned the Accession NumberIFO 15773.

[0458] The mouse pancreatic β cell line, designated MIN6, is on depositunder the terms of the Budapest Treaty from Dec. 27, 1994, with NIBH andhas been assigned the Accession Number FERM BP-4954. It is also ondeposit from Apr. 11, 1995 with IFO and has been assigned the AccessionNumber IFO 50454.

[0459] The transformant Escherichia coli, designated SURE/pTS863, whichis obtained in the Example 12 mentioned herein below, is on depositunder the terms of the Budapest Treaty from May 25, 1995, with NIBH andhas been assigned the Accession Number FERM BP-5110. It is also ondeposit from Jun. 1, 1995 with IFO and has been assigned the AccessionNumber IFO 15826.

[0460] The transformant CHO cell, designated CHO/pTS863-5, which isobtained in the Example 13 mentioned herein below, is on deposit underthe terms of the Budapest Treaty from May 25, 1995, with NIBH and hasbeen assigned the Accession Number FERM BP-5111. It is also on depositfrom Jun. 1, 1995 with IFO and has been assigned the Accession NumberIFO 50456.

[0461] The transformant CHO cell, designated CHO/pTS863-7, which isobtained in the Example 13 mentioned herein below, is on deposit underthe terms of the Budapest Treaty from May 25, 1995, with NIBH and hasbeen assigned the Accession Number FERM BP-5112. It is also on depositfrom Jun. 1, 1995 with IFO and has been assigned the Accession NumberIFO 50457.

[0462] Each SEQ ID NO set forth in the SEQUENCE LISTING of thespecification refers to the following sequence:

[0463] [SEQ ID NO: 1] is a partial amino acid sequence encoded by themouse pancreatic β-cell line, MIN6-derived galanin receptor protein cDNAincluded in p3H2-34,

[0464] [SEQ ID NO: 2] is a full length amino acid sequence encoded bythe mouse pancreatic β-cell line, MIN6-derived galanin receptor proteincDNA included in pMGR20,

[0465] [SEQ ID NO: 3] is a nucleotide sequence of the mouse pancreaticβ-cell line, MIN6-derived galanin receptor protein cDNA fragmentincluded in p3H2-34,

[0466] [SEQ ID NO: 4] is a nucleotide sequence of the translational unitin the mouse pancreatic β-cell line, MIN6-derived galanin receptorprotein cDNA fragment included in pMGR20,

[0467] [SEQ ID NO: 5] is a full length amino acid sequence encoded bythe human galanin receptor protein cDNA obtained in Example 11.

[0468] [SEQ ID NO: 6] is a nucleotide sequence of the translational unitin the human galanin receptor protein cDNA obtained in Example 11.

[0469] The practice of the present invention will employ, otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, pharmacology, immunology, bioscience, and medicaltechnology, which are within the skill of the art. All patents, patentapplications, and publications mentioned herein, both supra and infra,are hereby incorporated herein by reference.

EXAMPLES

[0470] Described below are working examples of the present inventionwhich are provided only for illustrative purposes, and not to limit thescope of the present invention. In light of the present disclosure,numerous embodiments within the scope of the claims will be apparent tothose of ordinary skill in the art. Incidentally, the gene operationusing Escherichia coli is carried out by a method described in Maniatis,et al.: “Molecular Cloning” (Cold Spring Harbor Laboratory, 1989).

Reference Example 1 Preparation of Synthetic DNA Primer for AmplifyingDNA Coding for G Protein Coupled Receptor Protein

[0471] A comparison of deoxyribonucleotide sequences coding for theknown amino acid sequences corresponding to or near the firstmembrane-spanning domain each of human-derived TRH receptor protein(HTRHR), human-derived RANTES receptor protein (L10918, HUMRANTES),human Burkitt's lymphoma-derived unknown ligand receptor protein(X68149, HSBLR1A), human-derived somatostatin receptor protein (L14856,HUMSOMAT), rat-derived μ-opioid receptor protein (U02083, RNU02083),rat-derived κ-opioid receptor protein (U00442, U00442), human-derivedneuromedin B receptor protein (M73482, HUMNMBR), human-derivedmuscarinic acetylcholine receptor protein (X15266, HSHM4), rat-derivedadrenaline α₁B receptor protein (L08609, RATAADRE01), human-derivedsomatostatin 3 receptor protein (M96738, HUMSSTR3X), human-derived C₅areceptor protein (HUMC5AAR), human-derived unknown ligand receptorprotein (HUMRDC1A), human-derived unknown ligand receptor protein(M84605, HUMOPIODRE) and rat-derived adrenaline α₂B receptor protein(M91466, RATA2BAR) was made. As a result, highly homologous regions orparts were found.

[0472] Further, a comparison of deoxynucleotide sequences coding for theknown amino acid sequences corresponding to or near the sixthmembrane-spanning domain each of mouse-derived unknown ligand receptorprotein (M80481, MUSGIR), human-derived bombesin receptor protein(L08893, HUMBOMB3S), human-derived adenosine A2 receptor protein(S46950, S46950), mouse-derived unknown ligand receptor protein (D21061,MUSGPCR), mouse-derived TRH receptor protein (S43387, S43387),rat-derived neuromedin K receptor protein (J05189, RATNEURA),rat-derived adenosine A1 receptor protein (M69045, RATALARA),human-derived neurokinin A receptor protein (M57414, HUMNEKAR),rat-derived adenosine A3 receptor protein (M94152, RATADENREC),human-derived somatostatin 1 receptor protein (M81829, HUMSRI1A),human-derived neurokinin 3 receptor protein (S86390, S86371S4),rat-derived unknown ligand receptor protein (x61496, RNCGPCR),human-derived somatostatin 4 receptor protein (L07061, HUMSSTR4Z) andrat-derived GnRH receptor protein (M31670, RATGNRHA) was made. As aresult, highly homologous regions or parts were found.

[0473] The aforementioned abbreviations in the parentheses areidentifiers (reference numbers) which are indicated when GenBank/EMBLData Bank is retrieved by using DNASIS Gene/Protein Sequencing Data Base(CD019, Hitachi Software Engineering, Japan) and are usually called“Accession Numbers” or “Entry Names”. HTRHR is, however, the sequence asdisclosed in Japanese Unexamined Patent Publication No. 286986/1993 (EPA638645).

[0474] Specifically, it was planned to incorporate mixed bases relyingupon the base regions that were in agreement with cDNAs coding for alarge number of receptor proteins in order to enhance base agreement ofsequences with as many receptor cDNAs as possible even in other regions.Based upon these sequences, the degenerate synthetic DNA having anucleotide sequence represented by SEQ ID NO: 7 which is complementaryto the homologous nucleotide sequence and the degenerate synthetic DNAhaving a nucleotide sequence represented by SEQ ID NO: 8 which iscomplementary to the homologous nucleotide sequence were produced.Nucleotide synthesis was carried out by a DNA synthesizer.

Synthetic DNAs

5′-CGTGG (G or C) C (A or C) T (G or C) (G or C) TGGGCAAC (A, G, C or T)(C or T) CCTG-3′  (SEQ ID NO: 7)

5′-GT (A, G, C or T) G (A or T) (A or G) (A or G) GGCA (A, G, C or T)CCAGCAGA (G or T) GGCAAA-3′  (SEQ ID NO: 8)

[0475] The parentheses indicate the incorporation of a plurality ofbases, leading to multiple oligonucleotides in the primer preparation.In other words, nucleotide residues in parentheses of the aforementionedDNAs were incorporated in the presence of a mixture of plural bases atthe time of synthesis.

Example 1 Preparation of Poly(A)⁺ RNA Fraction from Mouse Pancreaticβ-Cell Strain, MIN6 and Synthesis of cDNA

[0476] A total RNA was prepared from the mouse pancreatic β-cell strain,MIN6 (Jun-ichi Miyazaki et al., Endocrinology, Vol. 127, No. 1,p.126-132) according to the guanidine thiocyanate method (Kaplan B. B.et al., Biochem. J., 183, 181-184 (1979)) and, then, poly(A)⁺ RNAfractions were prepared with a mRNA purifying kit (Pharmacia Co.). Next,to 5 μg of the poly(A)⁺ RNA fraction was added a random DNA hexamer (BRLCo.) as a primer, and the resulting mixture was subjected to reactionwith mouse Moloney Leukemia virus (MMLV) reverse transcriptase (BRL Co.)in the buffer attached to the MMLV reverse transcriptase kit tosynthesize complementary DNAs. The reaction product was extracted withphenol/chloroform (1:1), precipitated in ethanol, and was then dissolvedin 30 μl of TE buffer (10 mM Tris-HCl at pH8.0, 1 mM EDTA at pH8.0).

Example 2 Amplification of Receptor cDNA by PCR Using MIN6-Derived cDNAand Sequencing

[0477] By using, as a template, 5 μl of cDNA prepared from the mousepancreatic β-cell strain, MIN6 in Example 1, PCR amplification using theDNA primers synthesized in Reference Example 1 was carried out. Areaction solution was composed of the synthetic DNA primers each in anamount of 100 pM, 0.25 mM dNTPs, 1 μl of Taq DNA polymerase and 10 μl of10×buffer attached to the enzyme kit, and the total amount of thereaction solution was made to be 100 μl. The cycle for amplificationincluding 96° C. for 30 sec., 45° C. for 1 min. and 60° C. for 3 min.was repeated 30 times by using a Thermal Cycler (Perkin-Elmer Co.).Prior to adding Taq DNA polymerase, the remaining reaction solution wasmixed and was heated at 95° C. for 5 minutes and at 65° C. for 5minutes. The amplified products were confirmed relying upon 1.2% agarosegel electrophoresis and ethidium bromide staining.

Example 3 Subcloning of PCR Product into Plasmid Vector and Selection ofNovel Receptor Candidate Clone via Decoding Nucleotide Sequence ofInserted cDNA Region

[0478] The PCR products obtained in Example 2 were separated with a 0.8%low-melting temperature agarose gel, the band parts were excised fromthe gel with a razor blade, and were heat-melted, extracted with phenoland precipitated in ethanol to recover DNAs. According to the protocolattached to a TA Cloning Kit (Invitrogen Co.), the recovered DNAs weresubcloned to the plasmid vector, pCR™ II (Invitrogen Co.). Therecombinant vectors were introduced into E. coli JM109 competent cells(Takara Shuzo Co., Japan) to produce transformants. Then, transformantclones having a cDNA-inserted fragment were selected in an LB(Luria-Bertani) agar culture medium containing ampicillin, IPTG(isopropylthio-β-D-galactoside) and X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside). Only transformant clonesexhibiting white color were picked with a sterilized toothstick toobtain transformant Escherichia coli JM109/p3H2-34.

[0479] The individual clones were cultured overnight in an LB culturemedium containing ampicillin and treated with an automatic plasmidextracting machine (Kurabo Co., Japan) to prepare plasmid DNAs. Analiquot of the DNAs thus prepared was cut by EcoRI to confirm the sizeof the cDNA fragment that was inserted. An aliquot of the remaining DNAswas further processed with RNase, extracted with phenol/chloroform, andprecipitated in ethanol so as to be condensed. Sequencing was carriedout by using a DyeDeoxy terminator cycle sequencing kit (ABI Co.), theDNAs were decoded by using a fluorescent automatic sequencer, and thedata of the nucleotide sequences obtained were read by using DNASIS(Hitachi System Engineering Co., Japan).

[0480] Homology retrieval was carried out based upon the determinednucleotide sequence [FIG. 1]. As a result, it was learned that a novel Gprotein coupled receptor protein was encoded by the cDNA fragment insertin the plasmid possessed by the transformant Escherichia coliJM109/p3H2-34. To further confirm this fact, by using DNASIS (HitachiSystem Engineering Co., Japan) the nucleotide sequence were convertedinto an amino acid sequence [FIG. 1], and homology retrieval was carriedout in view of hydrophobicity plotting [FIG. 2] and at the amino acidsequence level to find homology relative to human somatostatin receptorsubtype 4 (JN0605), human somatostatin receptor subtype 2 (B41795) andrat-derived ligand unknown receptor (A39297) [FIG. 3]. Abbreviations inparentheses are reference numbers assigned when they are registered asdata to NBRF-PIR/Swiss-PROT and are usually called “Accession Numbers”or “Entry Names”.

Example 4 Cloning of cDNA containing Whole Coding Region for ReceptorProtein from Mouse Pancreatic β-Cell Strain, MIN6-Derived cDNA Library

[0481] Superscript™ Lambda System (BRL, Cat. 8256) distributed by BRLCo. and Gigapack II Gold (Stratagene, Cat. 200215) distributed byStratagene Co. were used to construct MIN6-derived cDNA libraries. Byusing the above kits, a MIN6 cDNA library with 2.2×10⁶ pfu (plaqueforming units) was constructed from 10 μg of MIN6 poly(A)⁺ RNA. The cDNAlibrary was mixed with E. coli Y1090r⁻ treated with magnesium sulfate,and incubated at 37° C. for 15 minutes followed by addition of 0.5%agarose (Pharmacia Co.) LB. The E. coli was plated onto a 1.5% agar(Wako Pure Chemical Co., Japan) LB plate (containing 50 μg/ml ofampicillin). A nitrocellulose filter was placed on the plate on whichplaques were formed and the plaque was transferred onto the filter. Thefilter was denatured with an alkali and then heated at 80° C. for 3hours to fix DNAs.

[0482] The filter was incubated overnight at 42° C. together with theprobe mentioned herein below in a buffer containing 50% formamide,4×SSPE(20×SSPE (pH 7.4) is 3 M NaCl, 0.2 M NaH₂PO4.H₂O, 25 mM EDTA),5×Denhardt's solution, 0.1% SDS and 100 μg/ml of salmon sperm DNA forhybridization.

[0483] The probe used was obtained by cutting the DNA fragment insertedin the plasmid, p3H2-34, obtained in Example 2, with EcoRI, followed byrecovery and labeling by incorporation of [³²P]dCTP (Dupont/NEN) with arandom prime DNA labelling kit (Amersham Co.).

[0484] It was washed with 2×SSC (150 mM NaCl and 15 mM sodium citrate),0.1% SDS at 55° C. for 1 hour and, then, subjected to an autoradiographyat −80° C. to detect hybridized plaques.

[0485] In this screening, hybridization signals were recognized in twoindependent plaques. Each DNA was prepared from the two clones. The DNAsdigested with SalI and NotI were subjected to an agarose electrophoresisand were analyzed. Inserted fragments were identified at about 2.0 kband 3.0 kb, respectively. Between them, the DNA fragment correspondingto the band at about 3.0 kb (λ No.20) was selected. The λ No.20-derivedNotI-SalI fragment with about 3.0 kb was subcloned into the NotI-SalIsite of the plasmid, pBluescript™ II SK(+), and E. coli JM109 wastransformed with the plasmid to obtain a transformant E. coliJM109/pMGR20. A restriction enzyme map of the plasmid, pMGR20, wasprepared relying upon a restriction enzyme map deduced from thenucleotide sequence as shown in Example 2. As a result, it was learnedthat it carried a full-length receptor protein-encoding DNA which waspredicted from the receptor protein-encoding DNA as shown in Example 2.

Example 5 Sequencing of MIN6-Derived Receptor Protein Full-Length cDNA

[0486] Among the NotI-SalI fragments inserted in the plasmid, pMGR20,obtained in Example 4, the nucleotide sequence with total 1607 bp,including not only a region that is considered to be a receptorprotein-coding region (ORF) but also a neighboring region thereof wassequenced. Concretely speaking, by utilizing restriction enzyme sitesthat exist in the NotI-SalI fragments, unnecessary parts were removed ornecessary fragments were subcloned in order to prepare template plasmidsfor analyzing the nucleotide sequence thereof. As for the nucleotidesequences of part of the regions, primers for sequencing weresynthesized based upon the nucleotide sequences that were determinedalready and used to make confirmation.

[0487] The reaction for determining the nucleotide sequence (sequencing)was carried out with a DyeDeoxy terminator cycle sequencing kit (ABICo.), the DNA was decoded with the fluorescent automatic sequencer (ABICo.), and the data of the nucleotide sequence obtained were analyzedwith DNASIS (Hitachi System Engineering Co., Japan).

[0488]FIG. 4 shows a nucleotide sequence around an open reading frame(ORF) of a mouse galanin receptor protein encoded by the cDNA insert inpMGR20. The nucleotide sequence of mouse galanin receptorprotein-encoding DNA corresponds to from the 481st to 1525th nucleotidesof the nucleotide sequence in FIG. 4. The amino acid sequence of thereceptor protein encoded by the DNA insert was that as represented bySEQ ID NO: 2 (FIG. 4). Since the amino acid sequence has 92% homology tothe human-derived galanin receptor protein at the amino acid sequencelevel, it was learned that the cDNA insert in the pMGR20 is amouse-derived galanin receptor protein-encoding cDNA.

Example 6 Galanin Receptor Binding Experiment using MIN6 Cell MembraneFraction.

[0489] (1) Preparation of Membrane Fractions from MIN6 Cells.

[0490] MIN6 cells were cultured by a known method (Endocrinology, vol.127, pages 126-132, 1990). Thus, the culturing was carried out in aDulbecco modified Eagle's medium containing 15% of fetal bovine serum,4.5 g/liter of glucose, 5 μl/liter of mercaptoethanol, 75 mg/ml ofpenicillin and 50 mg/ml of streptomycin in the presence of 5% carbondioxide gas. The cultured cells were washed with PBS containing EDTA andexfoliated from the culturing device. The exfoliated cells wererecovered by centrifugation and subjected to the following method ofpreparing the membrane fractions.

[0491] The recovered cells (about 2.5 ml) were suspended in a buffer forhomogenization (containing 10 mM of NaHCO₃, 5 mM of EDTA, 0.5 mM ofPMSF, 10 μg/ml of pepstatin, 20 μg/ml of leupeptin and 4 μg/ml of E-64;pH: 7.2) and homogenized with a polytron homogenizer (Kinematica) at23,000 rpm for one minute. The resulting homogenate was centrifuged in aHitachi RP24A rotor using a Hitachi High-Speed Centrifuging Machine(type: CR26H) at 5,000 rpm for ten minutes. The supernatant liquid aftercentrifugation was recovered and subjected to an ultracentrifugation ina Hitachi RP42 rotor using a Hitachi Ultracentrifugal Machine (type:SCP70H) at 30,000 rpm for one hour to give pellets. The resultingpellets were again suspended in a buffer for the homogenization andstored at −70° C. until its actual use.

[0492] (2) Receptor Binding Experiment using MIN6 Cell MembraneFractions.

[0493] The MIN6 cell membrane fractions prepared by the method of theabove-mentioned (1) were diluted with a buffer for the receptor bindingexperiment (containing 20 mM of Tris, 1 mM of EDTA, 0.03% of NaN , 0.1%of BSA, 0.05% of CHAPS, 0.5 mM of PMSF, 10 μg/ml of pepstatin, 20 μg/mlof leupeptin and 4 μg/ml of E-64; pH: 7.4) to make the membrane proteinconcentration 50 μg/ml. Each 100 μl of the diluted membrane fractionswas charged in a test tube made of polypropylene (Falcon; type 2038) andsubjected to the following receptor binding experiment. In themeanwhile, porcine galanin (New England Nuclear) which was labeled witha commercially available [¹²⁵I] radioisotope was diluted with a bufferfor the receptor binding experiment to make its concentration 5 nM andused in the following experiments.

[0494] Standard porcine galanin solution or galanin-related peptidesolution (3 μl) with varied concentrations and 2 μl of 5 nM labeledgalanin solution were mixed with 100 μl of the above-mentioned membranefraction. The mixture was allowed to stand in a water bath of 25° C. for75 minutes to promote the receptor binding reaction. Thereafter, 1.5 mlof an ice-cooled buffer for the binding experiment was added to thereaction solution for quenching the binding reaction and filtered with aglass fiber filter (GF/F, manufactured by Whatman) immediately whereuponthe membrane fractions were collected on the filter paper. Then thefilter paper was washed with 1.5 ml of the same buffer and the amount ofthe radioisotope in the filter paper was determined by a gamma-raydetector.

[0495] Amount of the labeled galanin bound therewith was expressed interms of PMB (percent of maximum binding) as calculated by the followingequation (2):

PMB=[(B−NSB)/(B ₀ −NSB)]×100  (2)

[0496] in which PMB: percent of maximum binding

[0497] B: the value when the sample is added

[0498] NSB: nonspecific binding amount (the binding amount of thelabeled galanin in the presence of 1 μM of standard galanin)

[0499] B₀: maximum binding amount (the binding amount of the labeledgalanin in the absence of the standard porcine galanin)

[0500] The result wherein the binding amounts (PMB) of the labeledgalanin as determined hereinabove were plotted against theconcentrations of standard porcine galanin, rat galanin, galanin (1-16)partial peptide or galanin antagonist (galantide) is shown in FIG. 7.From the result, each of the concentrations (IC₅₀) giving 50% of PMB wascalculated and given in Table 1. TABLE 1 Peptides IC₅₀ Porcine Galanin0.12 nM Rat Galanin 0.13 nM Galanin (1-16)  3.3 nM Galantide 0.69 nM

[0501] It is noted from Table 1 that, when the cell membrane fractionsof MIN6 cells were used, the receptor binding affinity of the ligand tothe galanin receptor was able to be determined by means of a galaninreceptor binding experiments.

Example 7 Screening of Galanin Receptor Agonist/Antagonist by GalaninReceptor Binding Experiments

[0502] It is possible to conduct a screening of galanin receptorangonist/antagonist by a method mentioned in Example 6. Thus, 1 μl ofthe solution of test compounds and 2 μl of a 5 nM labeled galaninsolution are mixed with 100 μl of MIN6 cell membrane fractions by thesame manner as in Example 6. Thereafter, the binding amount isdetermined by the same manner as in Example 6 whereby the compoundswhich decreased the PMB to an extent of lower than a certain level arescreened as galanin receptor agonists/antagonists.

Example 8 Detection for Biological Activity of Galanin Using MIN6 Cells

[0503] It has been known that galanin is biologically active ininhibiting the insulin secretion of a Langerhans islet of pancreas bystimulation of glucose. Such an activity can be easily detected by thefollowing methods using MIN6 cells. Among the detecting methods,measurement of insulin secretion by glucose stimulation was principallyconducted according to a known method (Diabetologia, volume 36, pages1139-1145, 1993).

[0504] Briefly, 3×10⁵ MIN6 cells were seeded on a 24-well plate andcultured in a Dulbecco modified Eagle's medium (supplemented with 15% offetal bovine serum, 4.5 g/liter of glucose, 5 μl/liter ofmercaptoethanol, 75 mg/ml of penicillin and 50 mg/ml of streptomycin) inthe presence of 5% carbon dioxide gas for three days.

[0505] The cells were washed for three times with a Krebs-Ringer-HEPESbuffer (containing 119 mM of NaCl, 4.74 mM of KCl, 2.54 mM of CaCl₂,1.19 mM of MgSO₄, 1.19 mM of KH₂PO₄, 25 mM of NaHCO₃, 10 mM of HEPES and0.5% of BSA) and cultured in a Krebs-Ringer-HEPES buffer to which 5 mMof glucose was added at 37° C. for 30 minutes.

[0506] Then the cells were washed with the Krebs-Ringer-HEPES buffertwice. The cells were cultured at 37° C. for 90 minutes in aKrebs-Ringer-HEPES buffer to which a varied amount of rat galanin and 25mM of glucose. The supernatant liquid after the culturing was collectedand the amount of insulin which was secreted into the supernatant liquidwas determined by a commercially available radioimmunoassay kit(Amersham).

[0507]FIG. 8 shows the result in which the amount of insulin secretionwas plotted against the amount of galanin. It is apparent from FIG. 8that, when about 100 pM of galanin is added, the insulin secretion whichincreased by a glucose stimulation is decreased to an extent of aboutone half. As such, it is now clear that the activity of galanin can beeasily detected using the above-mentioned method.

Example 9 Detection for Biological Activity of Galanin Using MIN6 Cells

[0508] When MIN6 cells were treated with forskolin, the cAMPconcentration in the cells increased whereupon secretion of insulinincreased. Such a phenomenon can be inhibited by addition of galanin andcan be used as a method of measuring the biological activity of galanin.

[0509] The cells which were cultured and pretreated (by the operationsof washing, treating with 5 mM of glucose and washing) by the samemanner as in Example 8 were cultured at 37° C. for 90 minutes in aKrebs-Ringer-HEPES buffer to which varied concentration of rat-typegalanin, 100 μM of forskolin, 200 μM of isobutylmethylxantine (IBMX) and6.25 mM of glucose were added.

[0510] The supernatant after the culturing was recovered and the amountof insulin which was secreted into the supernatant was determined by acommercially available radioimmunoassay kit (Amersham). In addition, thecells were treated with perchloric acid to extract the cAMP in the cellsand the cAMP in the extract was determined by a commercially availableradioimmunoassay kit (Amersham). FIG. 9 shows the result in which thesecreted amount of insulin is plotted against the amount of galanin.FIG. 10 shows the result in which the amount of intracellular cAMP wasplotted. From FIG. 9 and FIG. 10, it is noted that the activity ofgalanin can be easily detected when secretion of insulin from MIN6 cellsor cAMP concentration in MIN6 cells was measured.

Example 10 Detection for Biological Activity of GalaninAgonist/Antagonist Using MIN6 Cells

[0511] It is possible to measure the activity of galaninagonist/antagonist by a method as mentioned in Example 8. Briefly, thecells which are cultured and pretreated (by the operations of washing,treating with 5 mM of glucose and washing) by the same manner as inExample 8 are cultured at 37° C. for 90 minutes in a Krebs-Ringer-HEPESbuffer to which a suitable concentration of test compounds, 100 pM ofrat galanin and 25 mM of glucose are added.

[0512] The supernatant liquid after the culturing is recovered and theamount of insulin secreted into the supernatant liquid is determinedusing a commercially available radioimmunoassay kit (Amersham). As acontrol, the amount of insulin is determined by the same manner for thesupernatant liquid obtained after culturing in a Krebs-Ringer-HEPESbuffer containing 25 mM glucose only and also in a Krebs-Ringer-HEPESbuffer containing 100 pM of rat galanin and 25 mM of glucose. It isnoted from the result that the activity of galanin agonist/antagonistcan be easily measured using MIN6 cells.

Example 11 Cloning of cDNA Coding for Human Galanin Receptor Protein

[0513] Human melanoma Bowes cells were cultured using a DMEM medium withhigh concentrations of glucose containing 10% fetal bovine serum at 37°C. under the condition of 95% air/5% CO₂ and, when it became almostconfluent, total RNA was prepared by a guanidine-thiocyanate method.From the resulting total RNA was prepared a poly A⁺ RNA fraction by anoligo(dT) cellulose column. The poly A⁺ RNA (10 μg) was treated with arandom hexamer and a reverse transcriptase to synthesize asingle-stranded DNA and then treated with Escherichia coli DNApolymerase I and RNase H to synthesize a double-stranded DNA whereupon adouble-stranded cDNA was synthesized from poly A⁺ RNA. Thisdouble-stranded cDNA was blunt ended with a T4 DNA polymerase and thenEcoRI adapters were added thereto. The resulting double-stranded cDNAwherein both terminals were added with EcoRI adapters was subjected to agel filtration to remove cDNA of about 1,000 bp and less and thenphosphate group was introduced into the EcoRI adapters using a T4polynucleotide kinase.

[0514] Then this cDNA was incorporated into a λ gt11 EcoRI arm andsubjected to an in vitro packaging to prepare a cDNA library (averagechain length: about 1.6 kbp; rate of insertion: 98%) of melanoma Bowescells having about 1.5×10⁶ pfu in total. The λ phage of this cDNAlibrary was infected with Escherichia coli Y109r⁻ strain, seeded on eachof soft agar plates at about 1.8×10⁴ plaques each and incubatedovernight at 42° C. to form plaques. The plaques were transferred to anitrocellulose filter, successively treated with a modifying solution(0.5N sodium hydroxide and 1.5M sodium chloride), a neutralizingsolution (0.5M Tris-HCl (pH: 7.0) and 1.5M sodium chloride) and 3×SSC(20×SSC=3M sodium chloride and 0.3M sodium citrate), air-dried and bakedat 80° C. for three hours whereupon the phage DNA was immobilized on thenitrocellulose filter.

[0515] On the other hand, in order to obtain the cDNA fragments to beused as a probe, synthetic oligonucleotides {circle over (1)} and{circle over (2)} were synthesized based upon a base sequence of theknown human galanin receptor cDNA [Habert-Ortoll, E. et al., Proceedingsof the National Academy of Sciences of the U.S.A., 91, 9780-9783(1994)].

5′-TCCGTGGACCGCTACGTGGCCATCGTG-3′(SEQ ID NO: 9)  {circle over (1)}

[0516] It is a synthetic oligonucleotide containing a sense sequence of+388 to +414 (wherein the translation initiation site was named +1).

5′-GACTTATCACACATGAGTACAATTGGTTGATGG-3′(SEQ ID NO: 10)  {circle over(2)}

[0517] It is a synthetic oligonucleotide containing an antisensesequence of +1024 to +1053.

[0518] An RT-PCR was carried out using those two synthetic nucleotidesas primers and 5 μg of human melanoma Bowes cell poly A⁺ RNA as atemplate whereupon cDNA fragments of 669 bp containing C-terminals ofhuman galanin receptor protein were obtained. The cDNA fragments wereincorporated into the HincII site of pUC119 to give a plasmid pHGR54-7.The pHGR54-7 was subjected to a double digestion with BamHI and HincIIand the resulting cDNA fragments containing the human galanin receptorprotein C-terminal were used as probes for screening the human melanomaBowes cell cDNA library.

[0519] Labeling of the probes was conducted by subjecting theabove-mentioned cDNA fragments to a random priming method using[α-³²P]dCTP. A hybridization was carried out at 85° C. in a buffer forhybridization (5×SSPE, 5×Denhardt's solution, 100 μg/l thermallymodified salmon sperm DNA, 0.1% SDS) containing labeled probes. Thefilter was finally washed in 0.1×SSC, 0.1% SDS solution at 50° C. andsubjected to an autoradiography to detect the plaques which werehybridized with the probes.

[0520] After the phage DNA was extracted from the phage clone lambdaHGR2 obtained by that method, cDNA fragments were cut out by digestingwith a restriction enzyme EcoRI and inserted into the EcoRI sites of thepUC118 plasmid to give pHGR2-3. The base sequence of the cDNA fragmentsinserted thereinto was determined by a conventional method using[α-³²P]dCTP whereupon it was found that said cDNA fragment was composedof 1,882 bp (FIG. 11 and FIG. 12; SEQ ID NO: 6). There was onesubstitution with a base as compared with the base sequence of the humangalanin receptor cDNA which was reported already [Habert-Ortoll, E. etal., Proceedings of the National Academy of Sciences of the U.S.A., 91,9780-9783 (1994)]. Said substitution with a base was within atranslation domain and is accompanied by an amino acid substitution,i.e., ¹⁵Cys(TGT)→¹⁵Trp(TGG) (FIG. 11 and FIG. 12; SEQ ID NO: 6). Assuch, a plasmid pHGR2-3 containing the human galanin receptor DNAfragments was obtained.

Example 12 Construction of Expression Plasmid containing Human GalaninReceptor Protein cDNA

[0521] pAKKO-111 (shown as pA1-11 in FIG. 13) was used as an expressionvector. The pAKKO-111 was constructed as follows: Briefly, pTB1417according to Japanese Unexamined Patent Publication No. Hei-05/076385was treated with HindIII and ClaI to give DNA fragments of 1.4 kbcontaining SRα promoters and poly A added signals. Further, pTB348[Naruo, K. et al., Biochemical and Biophysical Research Communications,128, 256-264(1985)] was treated with ClaI and SalI to give DNA fragmentsof 4.5 kb containing dihydrofolate reductase (dhfr) genes. Those DNAfragments were blunt ended with a T4 polymerase and ligated by a T4ligase to construct pAKKO-111 plasmid.

[0522] Human galanin receptor cDNA expression plasmid was prepared by amethod as shown in FIG. 13 from the plasmids pHGR2-3 and pHGR54-7containing the human galanin receptor cDNA fragments obtained in Example11. First, pHGR2-3 was subjected to a double digestion using restrictionenzymes BamHI and MunI and the resulting DNA fragments of about 1,190 bpwere inserted between BamHI and MunI sites of pHGR54-7. In themeanwhile, pHGR2-3 was digested with NcoI, the resulting fragments of495 bp were blunt ended with DNA polymerase I Klenow fragments, SalIlinkers were added thereto, then subjected to a double digestion withAgeI and SacII and the resulting DNA fragments of 200 bp were insertedbetween XmaI and SacII sites of the above-prepared plasmid whereupon aplasmid pTS862 which contained the translation unit only of the humangalanin receptor cDNA was obtained.

[0523] Finally, SalI DNA fragments containing the translation domain ofthe human galanin receptor protein cDNA of about 1.0 kbp obtained bydigesting the plasmid pTS862 with SalI were introduced into the SalIsite of pAKKO-111 in a regular order to give a human galanin receptorprotein cDNA expression plasmid pTS863. This expression plasmid pTS863was introduced into Escherichia coli to give a transformant Escherichiacoli SURE/pTS863.

Example 13 Expression of Human Galanin Receptor Protein cDNA in CHO(dhfr⁻) Cells

[0524] Four kinds of CHO (dhfr⁻) cells (in which cell numbers werestepwisely changed within a range of 3×10⁴ to 1×10⁶ cells) were sowed onlaboratory dishes with 10 cm diameter and cultured for 24 hours with aHam's F12 medium containing 10% of fetal bovine serum. The human galaninreceptor cDNA expression plasmid pTS863 (1.5 μg) obtained in Example 12was transfected to the above-prepared cells by a calcium phosphatemethod. After 24 hours from the transfection, the medium was substitutedwith a DMEM medium containing 10% of dialyzed fetal bovine serum and thecells wherein the plasmid was incorporated in chromosomes were selected.Colonies of the selected cells were cloned to give two clones of cellstrains CHO/pTS863-5 and CHO/pTS863-7 which highly expressed the humangalanin receptor in a stable manner.

Example 14 Measurement of Human Galanin Receptor Activity of HumanGalanin Receptor Expression CHO Cells

[0525] Human galanin receptor expression CHO cells were seeded on a12-well plate, cultured at 37° C. under the condition of 95% air/5% CO₂using a DMEM medium containing 10% of dialyzed fetal bovine serum untila confluent was resulted and the medium was exchanged on one day beforethe binding experiment was done whereupon the human galanin receptorhaving an amino acid sequence having SEQ ID NO: 5 was expressed.

[0526] Then the binding experiment to [¹²⁵I] galanin (porcine) wasconducted as follows. First, the cells were washed twice with each 1 mlof a buffer for measuring a binding (Hanks solution containing 0.1% BSAand 0.05% of CHAPS) warmed at 37° C., said buffer for bindingmeasurement was sucked, then 0.5 ml of a buffer for binding measurementcontaining 100 pM of [¹²⁵I] galanin (porcine) was added and a bindingreaction was carried out for one hour at 37° C. under the condition of95% air/5% CO₂. After completion of the reaction, the buffer for thebinding measurement was removed and washed thrice with each 1 ml of abuffer for the binding measurement warmed at 37° C. The amount of [¹²⁵I]galanin (porcine) bound with the cells was measured by a gamma-counterafter removing the cells with 0.2N sodium hydroxide and was defined as atotal binding amount. Incidentally, the same operation was conductedafter adding 1 μM of unlabeled galanin (porcine) at the binding reactionand the amount of [¹²⁵I] galanin bound with the cells was defined as thenonspecific binding amount. The results are given in Table 2. TABLE 2Total Binding Nonspecific Binding Cell Strain No. Amt. (cpm) Amt (cpm)CHO/pTS863-5 50466.8 ± 502.9   1458 ± 100.1 CHO/pTS863-7 59158.6 ±2095.1 1962.4 ± 56.3 

[0527] It was confirmed from Table 2 that CHO/pTS863-5 and CHO/pTS863-7which are cell strains expressing the human galanin receptor protein ofthe present invention in a high and stable manner were capable ofspecifically binding with galanin which is a ligand.

Example 15 Saturation Binding Experiments and Scatchard Plot Analysiswith Human [¹²⁵I] Galanin in GAL5 Cell Membrane Fractions

[0528] CHO cells expressing human galanin receptor proteins (GAL5 cell,denoted by CHO/pTS863-5 in Example 13) were cultured in a DMEM mediumcontaining 10% dialyzed serum, 2 mM glutamine, penicillin andstreptomycin at 37° C. under the condition of 95% air/5% CO₂. The cellswere collected with a phosphate-buffered saline (PBS) containing 1 g/lEDTA, suspended in a buffer for homogenization (10 mM HEPES, 5 mM EDTA,0.03 % NaN₃, 10 μg/ml of pepstatin, 0.5 mM phenylmethylsulfonyl fluoride(PMSF), 20 μg/ml of E-64, 40 μg/ml of leupeptin, pH 7.3) and homogenizedwith a Polytron homogenizer. The resultant homogenates were centrifugedat 2,500 rpm for 10 min under 4° C. The resultant supernatant wasultracentrifuged at 30,000 rpm for 60 min under 4° C. Pellets weresuspended in a buffer for homogenization to form a suspension as a GAL5cell membrane fraction.

[0529] The GAL5 cell membrane fraction was diluted with an assay buffer(20 mM Tris., 1 mM EDTA, 0.08 % NaN₃, 10 μg/ml of pepstatin, 0.5 mMPMSF, 20 μg/ml of E-64, 40 μg/ml of leupeptin, 0.1% BSA, and 0.05%CHAPS, pH 7.4) to make the membrane protein concentration 2 μg/ml. Each100 μl of the diluted membrane fractions was charged in a test tube.

[0530] The GAL5 cell membrane fractions were incubated with 15 pM to 500pM concentrations of human [¹²⁵I] galanin for 75 min at 25° C., thendiluted with 1.5 ml of a filtration buffer (20 mM Tris., 1 mM EDTA, 0.03% NaN₃, 0.1% BSA, and 0.05% CHAPS, pH 7.4, 4° C.) and subjected tofiltration through glass fiber filters (GF/F, Whatman, Kent, UK) treatedwith polyethylenimine. The filters were rinsed with 1.5 ml of the samefiltration buffer and the radiolabeled ligands remaining werequantitated with an auto-γ-counter (Beckman Instruments, Inc., PaloAlto, Calif.). Nonspecific binding was determined in the presence of 20nM of unlabeled human galanin. Scatchard plot analysis indicated itsdissociation constant (K_(d)) of 20 pM and maximal number of bindingsites (B_(MAX)) of 9.6 pmol/mg protein.

Example 16 Northern Blot Analysis Using Mouse Galanin Receptor ProteinEncoding cDNA

[0531] Five micrograms of poly (A)⁺ RNAs from mouse brain, thymus,spleen, lung, heart, liver, kidney, pancreas, testis, intestinal smoothmuscle, MIN6, and Neuro-2a were electrophoresed on 1.2% agarose gelafter denaturation with glyoxal and dimethyl sulfoxide (Thomas, P.S.,Proc. Natl. Acad. Sci. U.S.A., 77, 5201-5205, 1980). After theelectrophoresis, the RNAs were transferred onto a nitrocellulose filter(Schleicher & Schuell, Dassel, Germany) and the filter was baked at 80°C. for 2 hr. As a probe, the cDNA insert of p3H2-34 was excised by EcoRIdigestion and labeled with [α³²P]dCTP(222 TBq/mmol, Dupont/NEN) by aMultiprime DNA labeling kit (Amersham International PLC, Amersham Place,UK). Hybridization was conducted overnight at 42° C. in a buffercontaining 50% formamide, 5×SSC, 50 mM NaHPO₄, pH 6.5, 10×Denhardt'ssolution, and 100 μg/ml salmon sperm DNA. The filter was then washedwith 2×SSC, 0.1% SDS at 50° C., and then autoradiographed at −80° C. for12 days on an X-Omat film AR (Eastman Kodak Company, Rochester, N.Y.)with an intensifying screen. In Northern blot analysis using poly(A)RNAs from mouse tissues, the faintly hybridizing signals for the mousegalanin receptor only in the brain and small intestine (FIG. 14) weredetected . The result of the Northern blot indicated that the expressionlevel of galanin receptor mRNA was substantially lower in mouse normaltissues.

Example 17

[0532] (1) Expression of Mouse Galanin Receptor cDNA in CHO Cells

[0533] A cDNA clone with a complete translation unit, pMGR20 (obtainedin Example 4), was digested with NotI, blunt ended, and ligated withXbaI linker (Takara Shuzo Co., Ltd., Kyoto, Japan). The cDNA fragmentwas excised by SalI and XbaI digestion and inserted between SalI andSpeI sites of a mammalian cell expression vector, pAKKO-111H (Hinuma, etal., Biochim. Biophys. Acta, 1219, 251-259 (1994)). A resultantexpression plasmid with the mouse galanin receptor cDNA downstream ofthe SRα promoter and with dhfr gene as a selection marker was designatedas pAKKOMGR20. The plasmid DNA was transfected into CHO dhfr⁻ cells witha CellPhect Transfection Kit (Pharmacia). Transformants were selected inα-MEM medium without deoxyribonucleoside and ribonucleoside (GIBCO BRL)supplemented with dialyzed fetal bovine serum (GIBCQ BRL).

[0534] (2) Binding Assay with Porcine [¹²⁵I] Galanin

[0535] CHO cells transformed with pAKKOMGR20 and pAKKO-111H were grownin a 12-well tissue culture plate at 2.0×10⁵ cells/well and cultured forone day. After two washings with Hanks' balanced salt solution (HBSS)containing 0.1% BSA, the cells were incubated with 100 pM porcine [¹²⁵I]galanin (Dupont/NEN) at room temperature for 1 hour in the presence orabsence of unlabeled porcine galanin (1 μM at final concentration). Thecells were then washed three times with HBSS containing BSA, lysed with0.1N NaOH, 1% SDS, and the radiolabeled ligands remaining werequantitated with an auto-γ-counter (Beckman Instruments, Inc., PaloAlto, Calif.). For competitive binding experiments and Scatchard plotanalysis, membrane fractions were prepared from the transformed CHOcells. The cells grown in 225-cm² tissue culture flasks for three dayswere dispersed in a phosphate-buffered saline (PBS) containing 5 mM EDTAand then harvested by centrifugation. The cells were washed with thesame buffer, and then suspended in 10 mM sodium carbonate buffer (pH7.5), including 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF),20 μg/ml of leupeptin, 4 μg/ml of E-64, and 0.5 μg/ml of pepstatin.After the cells were homogenized with a Polytron homogenizer, thehomogenates were centrifuged at 3,000 rpm for 10 min in a Hitachi RR2A2rotor. The resultant supernatant was ultracentrifuged at 30,000 rpm for60 min in a Beckman Type 30 rotor. Pellet suspension was then done in abuffer containing 20 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.5 mM PMSF, 20μg/ml of leupeptin, 4 μg/ml of E-64, and 0.5 μg/ml of pepstatin, andused as a membrane fraction.

[0536] Competitive binding and saturation binding experiments wereperformed as described by Ohtaki et al. (J. Biol. Chem., 268,26650-26657, (1993)).

[0537] In brief, the membrane fractions were incubated in a buffercontaining 20 mM Tris-HCl (pH 7.5), 0.05% CHAPS, 0.1% BSA, 5 mM EDTA,0.5 mM of PMSF, 20 μg/ml of leupeptin, 4 μg/ml of E-64, and 0.5 μg/ml ofpepstatin with porcine [¹²⁵I] galanin at 25° C. for 75 min. Bound andfree ligands were separated by filtration through glass fiber filters(GF/F, Whatman, Kent, UK) treated with 0.3% of polyethylenimine.Nonspecific binding was determined in the presence of 1 μM of unlabeledporcine galanin. In competitive binding experiments the concentrationsof galanin and galanin analogs were added to the buffer simultaneouslywith porcine [¹²⁵I] galanin.

[0538] CHO cells transformed with the expression plasmid for the mousegalanin receptor cDNA bound significant amounts of [¹²⁵I] galanin ascompared with control cells (FIG. 15).

[0539] Membrane fractions (1 μg of protein) were incubated withconcentrations of porcine [¹²⁵I] galanin for 75 min at 25° C. inScatchard plot analysis. The results shown are from one representativeexperiment performed in triplicate assays. Each symbol represents themean value±S.E.M. B, [¹²⁵I] galanin bound (pmol/mg protein), B/F, boundto free ratio (pmol/mg protein•nM). Scatchard plot analysis indicatedthe presence of a single class of high-affinity binding site with adissociation constant (K_(d)) of 45 pM and maximal number of bindingsites (B_(MAX)) of 5 pmol/mg protein (FIG. 17).

[0540] Competitive experiments on the binding of porcine [¹²⁵I] galaninto mouse galanin receptor were conducted. Competitions to the porcine[¹²⁵I] galanin (100 pM at final concentrations) bindings were examinedwith unlabeled porcine (Δ), rat (), human (▪) galanins, galanin-(1-16)(◯), and M15 (▴). Membrane fractions (1 μg of protein) were incubatedwith the ligands for 75 min at 25° C. The amounts of [¹²⁵I] galaninbound were expressed as percentages against the control. Each symbolrepresents the mean value±S.E.M. of the triplicate assays. IC₅₀ valueswere 0.25±0.03 nM (porcine galanin), 0.25±0.01 nM (rat galanin),0.43±0.03 nM (human galanin), 0.83±0.01 nM (Ml5), and 3.6±0.04 nM[galanin-(1-16)], respectively.

[0541] The binding of [¹²⁵I] galanin was competitively inhibited bygalanin and galanin-derived peptides. Porcine and rat galanin exhibitedalmost the same high efficiency in inhibiting the [¹²⁵I] galanin bindingwhereas human galanin was somewhat lower. The Ki values of porcine, rat,and human galanins were 0.072±0.008, 0.069±0.002, and 0.12±0.008 nM,respectively. The galanin receptor antagonists M15 and galanin-(1-16)also effectively inhibit the [¹²⁵I] galanin binding, and their Ki valueswere 0.23±0.003 and 1.0±0.011 nM, respectively (FIG. 18). These obtainedvalues were almost comparable to those from MIN6 cell membranes.

[0542] (3) cAMP Assay

[0543] The CHO cells were seeded at 2.0×10⁵ cells/well in 24-well tissueculture plates and cultured for two days. The cells were washed twotimes with HBSS containing 0.1% BSA and 1 mM IBMX, and then the samebuffer with experimental agents were added to the wells. Afterincubation at 37° C. for 30 min, the media were discarded andintracellular cAMP was extracted with ice-cold ethanol. The aliquots ofextracts were evaporated and the amounts of cAMP were quantitated by acAMP EIA system (Amersham) as described by the manufacturer.

[0544] Galanin receptor-mediated inhibition of forskolin-stimulated cAMPproduction was observed. CHO-MGR20 or mock transformed CHO cells wereincubated with forskolin (10 μM) and porcine galanin (0.1 μM) at 37° C.for 30 min. The reaction was terminated by extracting the cells withice-cold ethanol. The amounts of intracellular cAMP were quantitated byEIA. Values indicated are mean±S.E.M. in triplicate assays.

[0545] It is examined by the assessment of galanin-induced signaltransduction to confirm further that the mouse galanin receptorexpressed in CHO cells was functional. The pancreatic galanin receptorhas been demonstrated to induce the inhibition of insulin releasethrough a pathway involving G proteins negatively coupled to adenylatecyclase (Cormont, M.,et al., Diabates, 40, 1170-1176, 1991; Gillison,S., et al., Diabates, 43, 24-32, 1994). The treatment with porcinegalanin potently inhibited forskolin-stimulated cAMP accumulation in thegalanin receptor cDNA-introduced CHO cells (FIG. 19). The CHOtransformants with the plasmid vector without cDNA insert also showedforskolin-stimulated cAMP accumulation, but it was not inhibited by thegalanin treatment. The treatment with galanin alone did not alter thecAMP levels in CHO transformants (FIG. 19).

[0546] The galanin receptor protein of the present invention and the DNAcoding for said protein can be used for {circle over (1)} acquisition ofantibody and antiserum; {circle over (2)} construction of expressionsystem for of a recombinant type receptor protein; {circle over (3)}development of the receptor binding assay system using said expressionsystem and screening of the candidate compounds for pharmaceuticals;{circle over (4)} conducting a drug design based upon a comparison withstructurally analogous ligands and receptors; {circle over (5)}preparation of probes and PCR primers for a gene diagnosis; {circle over(6)} preparation of transgenic animals; and {circle over (7)}preparation of model patient animals deficient in the receptor proteinDNA. Elucidation of the structure and property of the mouse-derivedgalanin receptor is particularly related to the development of uniquepharmaceuticals which act on such a system.

[0547] Furthermore, the human galanin receptor protein of the presentinvention is a novel protein having an amino acid sequence which isdifferent from that of the known human galanin receptor protein. Thecells (particularly CHO cells) retaining the expression vectorcontaining the human galanin receptor of the present invention arecapable of expressing far more amount of human galanin receptor proteinthan the known COS cells containing the human galanin receptor proteinare.

[0548] The human galanin receptor protein of the present invention orpartial peptide thereof or the cells containing the human galaninreceptor protein or a cell membrane fraction thereof is capable ofeffectively screening the human galanin receptor agonist or antagonist.

[0549] When the screening method of the present invention is used, it ispossible to advantageously select the agonist or the antagonist wherebypharmaceutical agents can be developed in earlier stage. The agonist isuseful, for example, as an inhibitor for acetylcholine liberation, aninhibitor for insulin secretion, an inhibitor for a learning behavior oran inhibitor for satiety and also as a preventive and therapeutic agentfor schizophrenic disease and as sedative while the antagonist isuseful, for example, as an accelerator for acetylcholine liberation, anaccelerator for insulin secretion, an inhibitor for growth hormonesecretion, an accelerator for a learning behavior or as an acceleratorfor satiety and also as a preventive and therapeutic agent for diabetes,Alzheimer's disease and dementia.

1 17 128 Amino acid Linear Peptide 1 Ala Ala Met Ser Val Asp Arg Tyr ValAla Ile Val His Ser Arg Arg 1 5 10 15 Ser Ser Ser Leu Arg Val Ser ArgAsn Ala Leu Leu Gly Val Gly Phe 20 25 30 Ile Trp Ala Leu Ser Ile Ala MetAla Ser Pro Val Ala Tyr His Gln 35 40 45 Arg Leu Phe His Arg Asp Ser AsnGln Thr Phe Cys Trp Glu Gln Trp 50 55 60 Pro Asn Lys Leu His Lys Lys AlaTyr Val Val Cys Thr Phe Val Phe 65 70 75 80 Gly Tyr Leu Leu Pro Leu LeuLeu Ile Cys Phe Cys Tyr Ala Lys Val 85 90 95 Leu Asn His Leu His Lys LysLeu Lys Asn Met Ser Lys Lys Ser Glu 100 105 110 Ala Ser Lys Lys Lys ThrAla Gln Thr Val Leu Val Val Val Val Val 115 120 125 348 Amino acidLinear Peptide 2 Met Glu Leu Ala Met Val Asn Leu Ser Glu Gly Asn Gly SerAsp Pro 1 5 10 15 Glu Pro Pro Ala Pro Glu Ser Arg Pro Leu Phe Gly IleGly Val Glu 20 25 30 Asn Phe Ile Thr Leu Val Val Phe Gly Leu Ile Phe AlaMet Gly Val 35 40 45 Leu Gly Asn Ser Leu Val Ile Thr Val Leu Ala Arg SerLys Pro Gly 50 55 60 Lys Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn LeuSer Ile Ala 65 70 75 80 Asp Leu Ala Tyr Leu leu Phe Cys Ile Pro Phe GlnAla Thr Val Tyr 85 90 95 Ala Leu Pro Thr Trp Val Leu Gly Ala Phe Ile CysLys Phe Ile His 100 105 110 Tyr Phe Phe Thr Val Ser Met Leu Val Ser IlePhe Thr Leu Ala Ala 115 120 125 Met Ser Val Asp Arg Tyr Val Ala Ile ValHis Ser Arg Arg Ser Ser 130 135 140 Ser Leu Arg Val Ser Arg Asn Ala LeuLeu Gly Val Gly Phe Ile Trp 145 150 155 160 Ala Leu Ser Ile Ala Met AlaSer Pro Val Ala Tyr His Gln Arg Leu 165 170 175 Phe His Arg Asp Ser AsnGln Thr Phe Cys Trp Glu Gln Trp Pro Asn 180 185 190 Lys Leu His Lys LysAla Tyr Val Val Cys Thr Phe Val Phe Gly Tyr 195 200 205 Leu Leu Pro LeuLeu Leu Ile Cys Phe Cys Tyr Ala Lys Val Leu Asn 210 215 220 His Leu HisLys Lys Leu Lys Asn Met Ser Lys Lys Ser Glu Ala Ser 225 230 235 240 LysLys Lys Thr Ala Gln Thr Val Leu Val Val Val Val Val Phe Gly 245 250 255Ile Ser Trp Leu Pro His His Val Val His Leu Trp Ala Glu Phe Gly 260 265270 Ala Phe Pro Leu Thr Pro Ala Ser Phe Phe Phe Arg Ile Thr Ala His 275280 285 Cys Leu Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe290 295 300 Leu Ser Glu Asn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys CysHis 305 310 315 320 Val Cys Asp Glu Ser Pro Arg Ser Glu Thr Lys Glu AsnLys Ser Arg 325 330 335 Met Asp Thr Pro Pro Ser Thr Asn Cys Thr His Val340 345 384 Nucleic acid Double Linear cDNA S 3 GCCGCGATGT CTGTGGATCGCTACGTGGCC ATTGTGCACT CGCGGCGCTC CTCCTCCCTC 60 AGGGTGTCCC GCAACGCACTGCTGGGCGTG GGCTTCATCT GGGCGCTGTC CATCGCCATG 120 GCCTCGCCGG TGGCCTACCACCAGCGTCTT TTCCATCGGG ACAGCAACCA GACCTTCTGC 180 TGGGAGCAGT GGCCCAACAAGCTCCACAAG AAGGCTTACG TGGTGTGCAC TTTCGTCTTT 240 GGGTACCTTC TGCCCTTACTGCTCATCTGC TTTTGCTATG CCAAGGTCCT TAATCATCTG 300 CATAAAAAGC TGAAAAACATGTCAAAAAAG TCTGAAGCAT CCAAGAAAAA GACTGCACAG 360 ACCGTCCTGG TGGTCGTTGTAGTA 384 1044 Nucleic acid Double Linear cDNA S 4 ATGGAACTGG CTATGGTGAACCTCAGTGAA GGGAATGGGA GCGACCCAGA GCCGCCAGCC 60 CCGGAGTCCA GGCCGCTCTTCGGCATTGGC GTGGAGAACT TCATTACGCT GGTAGTGTTT 120 GGCCTGATTT TCGCGATGGGCGTGCTGGGC AACAGCCTGG TGATCACCGT GCTGGCGCGC 180 AGCAAACCAG GCAACCCCCGCAGCACCACC AACCTGTTTA TCCTCAATCT GAGCATCGCA 240 GACCTGGCCT ACCTGCTCTTCTGCATCCCT TTTCAGGCCA CCGTGTATGC ACTGCCCACC 300 TGGGTGCTGG GCGCCTTCATCTGCAAGTTT ATACACTACT TCTTCACCGT GTCCATGCTG 360 GTGAGCATCT TCACCCTGGCCGCGATGTCT GTGGATCGCT ACGTGGCCAT TGTGCACTCG 420 CGGCGCTCCT CCTCCCTCAGGGTGTCCCGC AACGCACTGC TGGGCGTGGG CTTCATCTGG 480 GCGCTGTCCA TCGCCATGGCCTCGCCGGTG GCCTACCACC AGCGTCTTTT CCATCGGGAC 540 AGCAACCAGA CCTTCTGCTGGGAGCAGTGG CCCAACAAGC TCCACAAGAA GGCTTACGTG 600 GTGTGCACTT TCGTCTTTGGGTACCTTCTG CCCTTACTGC TCATCTGCTT TTGCTATGCC 660 AAGGTCCTTA ATCATCTGCATAAAAAGCTG AAAAACATGT CAAAAAAGTC TGAAGCATCC 720 AAGAAAAAGA CTGCACAGACCGTCCTGGTG GTCGTTGTAG TATTTGGCAT ATCCTGGCTG 780 CCCCATCATG TCGTCCACCTCTGGGCTGAG TTTGGAGCCT TCCCACTGAC GCCAGCTTCC 840 TTCTTCTTCA GAATCACCGCCCATTGCCTG GCATACAGCA ACTCCTCAGT GAACCCCATC 900 ATATATGCCT TTCTCTCAGAAAACTTCCGG AAGGCGTACA AGCAAGTGTT CAAGTGTCAT 960 GTTTGCGATG AATCTCCACGCAGTGAAACT AAGGAAAACA AGAGCCGGAT GGACACCCCG 1020 CCATCCACCA ACTGCACCCACGTG 1044 349 Amino acid Linear Peptide 5 Met Glu Leu Ala Val Gly AsnLeu Ser Glu Gly Asn Ala Ser Trp Pro 1 5 10 15 Glu Pro Pro Ala Pro GluPro Gly Pro Leu Phe Gly Ile Gly Val Glu 20 25 30 Asn Phe Val Thr Leu ValVal Phe Gly Leu Ile Phe Ala Leu Gly Val 35 40 45 Leu Gly Asn Ser Leu ValIle Thr Val Leu Ala Arg Ser Lys Pro Gly 50 55 60 Lys Pro Arg Ser Thr ThrAsn Leu Phe Ile Leu Asn Leu Ser Ile Ala 65 70 75 80 Asp Leu Ala Tyr LeuLeu Phe Cys Ile Pro Phe Gln Ala Thr Val Tyr 85 90 95 Ala Leu Pro Thr TrpVal Leu Gly Ala Phe Ile Cys Lys Phe Ile His 100 105 110 Tyr Phe Phe ThrVal Ser Met Leu Val Ser Ile Phe Thr Leu Ala Ala 115 120 125 Met Ser ValAsp Arg Tyr Val Ala Ile Val His Ser Arg Arg Ser Ser 130 135 140 Ser LeuArg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Cys Ile Trp 145 150 155 160Ala Leu Ser Ile Ala Met Ala Ser Pro Val Ala Tyr His Gln Gly Leu 165 170175 Phe His Pro Arg Ala Ser Asn Gln Thr Phe Cys Trp Glu Gln Trp Pro 180185 190 Asp Pro Arg His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly195 200 205 Tyr Leu Leu Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys ValLeu 210 215 220 Asn His Leu His Lys Lys Leu Lys Asn Met Ser Lys Lys SerGlu Ala 225 230 235 240 Ser Lys Lys Lys Thr Ala Gln Thr Val Leu Val ValVal Val Val Phe 245 250 255 Gly Ile Ser Trp Leu Pro His His Ile Ile HisLeu Trp Ala Glu Phe 260 265 270 Gly Val Phe Pro Leu Thr Pro Ala Ser PheLeu Phe Arg Ile Thr Ala 275 280 285 His Cys Leu Ala Tyr Ser Asn Ser SerVal Asn Pro Ile Ile Tyr Ala 290 295 300 Phe Leu Ser Glu Asn Phe Arg LysAla Tyr Lys Gln Val Phe Lys Cys 305 310 315 320 His Ile Arg Lys Asp SerHis Leu Ser Asp Thr Lys Glu Asn Lys Ser 325 330 335 Arg Ile Asp Thr ProPro Ser Thr Asn Cys Thr His Val 340 345 1047 Nucleic acid Double LinearcDNA S 6 ATGGAGCTGG CGGTCGGGAA CCTCAGCGAG GGCAACGCGA GCTGGCCGGAGCCCCCCGCC 60 CCGGAGCCCG GGCCGCTGTT CGGCATCGGC GTGGAGAACT TCGTCACGCTGGTGGTGTTC 120 GGCCTGATCT TCGCGCTGGG CGTGCTGGGC AACAGCCTAG TGATCACCGTGCTGGCGCGC 180 AGCAAGCCGG GCAAGCCGCG GAGCACCACC AACCTGTTCA TCCTCAACCTGAGCATCGCC 240 GACCTGGCCT ACCTGCTCTT CTGCATCCCC TTCCAGGCCA CCGTGTACGCGCTGCCCACC 300 TGGGTGCTGG GCGCCTTCAT CTGCAAGTTC ATCCACTACT TCTTCACCGTGTCCATGCTG 360 GTGAGCATCT TCACCCTGGC CGCGATGTCC GTGGACCGCT ACGTGGCCATCGTGCACTCG 420 CGGCGCTCCT CCTCCCTCAG GGTGTCCCGC AACGCGCTGC TGGGCGTGGGCTGCATCTGG 480 GCGCTGTCCA TTGCCATGGC CTCGCCCGTG GCCTACCACC AGGGCCTCTTCCACCCGCGC 540 GCCAGCAACC AGACCTTCTG CTGGGAGCAG TGGCCCGACC CTCGCCACAAGAAGGCCTAC 600 GTGGTGTGCA CCTTCGTCTT CGGCTACCTG CTGCCGCTCC TGCTCATCTGCTTCTGCTAT 660 GCCAAGGTCC TTAATCACTT GCATAAAAAG TTGAAGAACA TGTCAAAGAAGTCTGAAGCA 720 TCCAAGAAAA AGACTGCACA GACAGTTCTG GTGGTGGTTG TGGTGTTTGGAATCTCCTGG 780 CTGCCGCACC ACATCATCCA TCTCTGGGCT GAGTTTGGAG TTTTCCCGCTGACGCCGGCT 840 TCCTTCCTCT TCAGAATCAC CGCCCACTGC CTGGCGTACA GCAATTCCTCCGTGAATCCT 900 ATCATTTATG CATTTCTCTC TGAAAATTTC AGGAAGGCCT ATAAACAAGTGTTCAAGTGT 960 CACATTCGCA AAGATTCACA CCTGAGTGAT ACTAAAGAAA ATAAAAGTCGAATAGACACC 1020 CCACCATCAA CCAATTGTAC TCATGTG 1047 25 Nucleic acidSingle Linear Other nucleic acid 7 CGTGGSCMTS STGGGCAACN YCCTG 25 27Nucleic acid Single Linear Other nucleic acid 8 GTNGWRRGGC ANCCAGCAGAKGGCAAA 27 27 Nucleic acid Single Linear Other nucleic acid 9 TCCGTGGACCGCTACGTGGC CATCGTG 27 33 Nucleic acid Single Linear Other nucleic acid10 GACTTATCAC ACATGAGTAC AATTGGTTGA TGG 33 1882 base pairs nucleic acidsingle linear Coding Sequence 722...1768 11 CGCGGATTTC AGCCGAGCTGTTTTCGCCTC TCAGTTGCAG CAGAGAAGCC CCTGGCACCC 60 GACTCTATCC ACCACCAGGAAGCCTCCCAA AAGAGCTCTC GCCCTGTGGA CGACTCGGAA 120 TCCCTGGAAA AGCCGGGAGGGAGTCGGAGG CGCCAGCCCA CTGGGGAGGT GGCGCTGGGC 180 GCGCGGGATG CGCGGGGAGCCTTCTCTGCA GGAGCCGCAC AGTGCACTGC TGCGCGCTGG 240 GCAGTGCGGG GAAGCGCCGCGGGAAGGAGC GGCTCCGAGC AACAGGTGCA GCACGCAGCC 300 GCTCCGGGAG CCAGGGAAAACCGCCGGCGA AGATCTGGAG CGGTAAGGCG GAGAGAAGGG 360 TCTTTCCACC TGCGCGGCTGCAGCCGGCGG ATCCCTCTTC CCAGGCTCCG TGGTCGCGCA 420 GCGGGCGGAG GCGCCCGGGCAGGGGACCCC CAGTGCTCTC GAGATCACCG TCCCTTCCCG 480 AGAAGGTCCA GCTCCGGGCTCCCGAACCCA CCCTCTCTCA GAAGGTCGCG GCGCAAAGAC 540 GGTGCCACCA GGCACGGCCACCGGATCCCC GCTCCCGCTG GCTCGCGCCT CGGGGGAAGC 600 TCAGACTCCT AAACTCGCACTCTCCGTGCT TTGCGCCGGG ACCCCTGGCC ACCCCCGGCG 660 CCTGCTATCC CGCCCTCCCTCCCCGCGCGC CCCGCCGCTC GCCGGGACAG CCCCGCGGGC 720 C ATG GAG CTG GCG GTCGGG AAC CTC AGC GAG GGC AAC GCG AGC TGG CCG 769 Met Glu Leu Ala Val GlyAsn Leu Ser Glu Gly Asn Ala Ser Trp Pro 1 5 10 15 GAG CCC CCC GCC CCGGAG CCC GGG CCG CTG TTC GGC ATC GGC GTG GAG 817 Glu Pro Pro Ala Pro GluPro Gly Pro Leu Phe Gly Ile Gly Val Glu 20 25 30 AAC TTC GTC ACG CTG GTGGTG TTC GGC CTG ATC TTC GCG CTG GGC GTG 865 Asn Phe Val Thr Leu Val ValPhe Gly Leu Ile Phe Ala Leu Gly Val 35 40 45 CTG GGC AAC AGC CTA GTG ATCACC GTG CTG GCG CGC AGC AAG CCG GGC 913 Leu Gly Asn Ser Leu Val Ile ThrVal Leu Ala Arg Ser Lys Pro Gly 50 55 60 AAG CCG CGG AGC ACC ACC AAC CTGTTC ATC CTC AAC CTG AGC ATC GCC 961 Lys Pro Arg Ser Thr Thr Asn Leu PheIle Leu Asn Leu Ser Ile Ala 65 70 75 80 GAC CTG GCC TAC CTG CTC TTC TGCATC CCC TTC CAG GCC ACC GTG TAC 1009 Asp Leu Ala Tyr Leu Leu Phe Cys IlePro Phe Gln Ala Thr Val Tyr 85 90 95 GCG CTG CCC ACC TGG GTG CTG GGC GCCTTC ATC TGC AAG TTC ATC CAC 1057 Ala Leu Pro Thr Trp Val Leu Gly Ala PheIle Cys Lys Phe Ile His 100 105 110 TAC TTC TTC ACC GTG TCC ATG CTG GTGAGC ATC TTC ACC CTG GCC GCG 1105 Tyr Phe Phe Thr Val Ser Met Leu Val SerIle Phe Thr Leu Ala Ala 115 120 125 ATG TCC GTG GAC CGC TAC GTG GCC ATCGTG CAC TCG CGG CGC TCC TCC 1153 Met Ser Val Asp Arg Tyr Val Ala Ile ValHis Ser Arg Arg Ser Ser 130 135 140 TCC CTC AGG GTG TCC CGC AAC GCG CTGCTG GGC GTG GGC TGC ATC TGG 1201 Ser Leu Arg Val Ser Arg Asn Ala Leu LeuGly Val Gly Cys Ile Trp 145 150 155 160 GCG CTG TCC ATT GCC ATG GCC TCGCCC GTG GCC TAC CAC CAG GGC CTC 1249 Ala Leu Ser Ile Ala Met Ala Ser ProVal Ala Tyr His Gln Gly Leu 165 170 175 TTC CAC CCG CGC GCC AGC AAC CAGACC TTC TGC TGG GAG CAG TGG CCC 1297 Phe His Pro Arg Ala Ser Asn Gln ThrPhe Cys Trp Glu Gln Trp Pro 180 185 190 GAC CCT CGC CAC AAG AAG GCC TACGTG GTG TGC ACC TTC GTC TTC GGC 1345 Asp Pro Arg His Lys Lys Ala Tyr ValVal Cys Thr Phe Val Phe Gly 195 200 205 TAC CTG CTG CCG CTC CTG CTC ATCTGC TTC TGC TAT GCC AAG GTC CTT 1393 Tyr Leu Leu Pro Leu Leu Leu Ile CysPhe Cys Tyr Ala Lys Val Leu 210 215 220 AAT CAC TTG CAT AAA AAG TTG AAGAAC ATG TCA AAG AAG TCT GAA GCA 1441 Asn His Leu His Lys Lys Leu Lys AsnMet Ser Lys Lys Ser Glu Ala 225 230 235 240 TCC AAG AAA AAG ACT GCA CAGACA GTT CTG GTG GTG GTT GTG GTG TTT 1489 Ser Lys Lys Lys Thr Ala Gln ThrVal Leu Val Val Val Val Val Phe 245 250 255 GGA ATC TCC TGG CTG CCG CACCAC ATC ATC CAT CTC TGG GCT GAG TTT 1537 Gly Ile Ser Trp Leu Pro His HisIle Ile His Leu Trp Ala Glu Phe 260 265 270 GGA GTT TTC CCG CTG ACG CCGGCT TCC TTC CTC TTC AGA ATC ACC GCC 1585 Gly Val Phe Pro Leu Thr Pro AlaSer Phe Leu Phe Arg Ile Thr Ala 275 280 285 CAC TGC CTG GCG TAC AGC AATTCC TCC GTG AAT CCT ATC ATT TAT GCA 1633 His Cys Leu Ala Tyr Ser Asn SerSer Val Asn Pro Ile Ile Tyr Ala 290 295 300 TTT CTC TCT GAA AAT TTC AGGAAG GCC TAT AAA CAA GTG TTC AAG TGT 1681 Phe Leu Ser Glu Asn Phe Arg LysAla Tyr Lys Gln Val Phe Lys Cys 305 310 315 320 CAC ATT CGC AAA GAT TCACAC CTG AGT GAT ACT AAA GAA AAT AAA AGT 1729 His Ile Arg Lys Asp Ser HisLeu Ser Asp Thr Lys Glu Asn Lys Ser 325 330 335 CGA ATA GAC ACC CCA CCATCA ACC AAT TGT ACT CAT GTG TGATAAAAGA TA 1780 Arg Ile Asp Thr Pro ProSer Thr Asn Cys Thr His Val 340 345 GAGTATCCTT ATGGTTGAGT TTCCATATAAGTGGACCAGA CACAGAAACA AACAGAATGA 1840 GCTAGTAAGC GATGCTGCAA CTTGTTATCTTAACAAGAAT TC 1882 145 amino acids amino acid single linear None 12 ValGly Leu Val Gly Asn Phe Leu Ala Ala Met Ser Val Asp Arg Tyr 1 5 10 15Val Ala Ile Val His Ser Arg Arg Ser Ser Ser Leu Arg Val Ser Arg 20 25 30Asn Ala Leu Leu Gly Val Gly Phe Ile Trp Ala Leu Ser Ile Ala Met 35 40 45Ala Ser Pro Val Ala Tyr His Gln Arg Leu Phe His Arg Asp Ser Asn 50 55 60Gln Thr Phe Cys Trp Glu Gln Trp Pro Asn Lys Leu His Lys Lys Ala 65 70 7580 Tyr Val Val Cys Thr Phe Val Phe Gly Tyr Leu Leu Pro Leu Leu Leu 85 9095 Ile Cys Phe Cys Tyr Ala Lys Val Leu Asn His Leu His Lys Lys Leu 100105 110 Lys Asn Met Ser Lys Lys Ser Glu Ala Ser Lys Lys Lys Thr Ala Gln115 120 125 Thr Val Leu Val Val Val Val Val Phe Ala Leu Cys Trp Leu ProPhe 130 135 140 Tyr 145 147 amino acids amino acid single linear None 13Met Phe Thr Ser Val Phe Cys Leu Thr Val Leu Ser Val Asp Arg Tyr 1 5 1015 Val Ala Val Val His Pro Leu Arg Ala Ala Thr Tyr Arg Arg Pro Ser 20 2530 Val Ala Lys Leu Ile Asn Leu Gly Val Trp Leu Ala Ser Leu Leu Val 35 4045 Thr Leu Pro Ile Ala Ile Phe Ala Asp Thr Arg Pro Ala Arg Gly Gly 50 5560 Gln Ala Val Ala Cys Asn Leu Gln Trp Pro His Pro Ala Trp Ser Ala 65 7075 80 Val Phe Val Val Tyr Thr Phe Leu Leu Gly Phe Leu Leu Pro Val Leu 8590 95 Ala Ile Gly Leu Cys Tyr Leu Leu Ile Val Gly Lys Met Arg Ala Val100 105 110 Ala Leu Arg Ala Gly Trp Gln Gln Arg Arg Arg Ser Glu Lys LysIle 115 120 125 Thr Arg Leu Val Leu Met Val Val Val Val Phe Val Leu CysTrp Met 130 135 140 Pro Phe Tyr 145 148 amino acids amino acid singlelinear None 14 Gln Phe Thr Ser Ile Phe Cys Leu Thr Val Met Ser Ile AspArg Tyr 1 5 10 15 Leu Ala Val Val His Pro Ile Lys Ser Ala Lys Trp ArgArg Pro Arg 20 25 30 Thr Ala Lys Met Ile Thr Met Ala Val Trp Gly Val SerLeu Leu Val 35 40 45 Ile Leu Pro Ile Met Ile Tyr Ala Gly Leu Arg Ser AsnGln Trp Gly 50 55 60 Arg Ser Ser Cys Thr Ile Asn Trp Pro Gly Glu Ser GlyAla Trp Tyr 65 70 75 80 Thr Gly Phe Ile Ile Tyr Thr Phe Ile Leu Gly PheLeu Val Pro Leu 85 90 95 Thr Ile Ile Cys Leu Cys Tyr Leu Phe Ile Ile IleLys Val Lys Ser 100 105 110 Ser Gly Ile Arg Val Gly Ser Ser Lys Arg LysLys Ser Glu Lys Lys 115 120 125 Val Thr Arg Met Val Ser Ile Val Val AlaAsx Phe Ile Phe Cys Trp 130 135 140 Leu Pro Phe Tyr 145 148 amino acidsamino acid single linear None 15 Met Phe Thr Ser Ile Tyr Cys Leu Thr ValLeu Ser Val Asp Arg Tyr 1 5 10 15 Val Ala Val Val His Pro Ile Lys AlaAla Arg Tyr Arg Arg Pro Thr 20 25 30 Val Ala Lys Val Val Asn Leu Gly ValTrp Val Leu Ser Leu Leu Val 35 40 45 Ile Leu Pro Ile Val Val Phe Ser ArgThr Ala Ala Asn Ser Asp Gly 50 55 60 Thr Val Ala Cys Asn Met Leu Met ProGlu Pro Ala Gln Arg Trp Leu 65 70 75 80 Val Gly Phe Val Leu Thr Tyr PheLeu Met Gly Phe Leu Leu Pro Val 85 90 95 Gly Ala Ile Cys Leu Cys Tyr CysLeu Ile Ile Ala Lys Met Arg Met 100 105 110 Val Ala Leu Lys Ala Gly TrpGln Gln Arg Lys Arg Ser Glu Arg Lys 115 120 125 Ile Thr Leu Met Val MetMet Val Val Met Val Phe Val Ile Cys Trp 130 135 140 Met Pro Phe Tyr 145436 base pairs nucleic acid single linear 16 GTGGGCCTGG TGGGCAACTTCCTGGCCGCG ATGTCTCGTG CATCGCTACG TGGCCATTGT 60 GCACTCGCGG CGCTCCTCCTCCCTCAGGGT GTCCCGCAAC GCACTGCTGG GCGTGGGCTT 120 CATCTGGGCG CTGTCCATCGCCATGGCCTC GCCGGTGGCC TACCACCAGC GTCTTTTCCA 180 TCGGGACAGC AACCAGACCTTCTGCTGGGA GCAGTGGCCC AACAAGCTCC ACAAGAAGGC 240 TTACGTGGTG TGCACTTTCGTCTTTGGGTA CCTTCTGCCC TTACTGCTCA TCTGCTTTTG 300 CTATGCCAAG GTCCTTAATCATCTGCATAA AAAGCTGAAA AACATGTCAA AAAAGTCTGA 360 AGCATCCAAG AAAAAGACTGCACAGACCGT CCTGGTGGTC GTTGTAGTAT TTGCCCTCTG 420 CTGGCTGCCT TTCTAC 4361607 base pairs nucleic acid single linear Coding Sequence 484...1524 17ATGGAACTGG CTATGGTGAA CCTCAGTGAA GGGAATGGGA GCGACCCAGA GCCGCCAGCC 60CCGGAGTCCA GGCCGCTCTT CGGCATTGGC GTGGAGAACT TCATTACGCT GGTAGTGTTT 120GGCCTGATTT TCGCGATGGG CGTGCTGGGC AACAGCCTGG TGATCACCGT GCTGGCGCGC 180AGCAAACCAG GCAACCCCCG CAGCACCACC AACCTGTTTA TCCTCAATCT GAGCATCGCA 240GACCTGGCCT ACCTGCTCTT CTGCATCCCT TTTCAGGCCA CCGTGTATGC ACTGCCCACC 300TGGGTGCTGG GCGCCTTCAT CTGCAAGTTT ATACACTACT TCTTCACCGT GTCCATGCTG 360CTGTCCTGGG CCACTCCGTG ATCCTAGGCT ACCTCCAGAG CCAGTTTTCC CTGGCTGGCA 420CAACTCTCCA GGGCGCTCCG GTCCGTTGCA CAGCGCCCCA AGGGGGTATC CCAGTAAGTG 480ATG GAA CTG GCT ATG GTG AAC CTC AGT GAA GGG AAT GGG AGC GAC CCA 528 GluLeu Ala Met Val Asn Leu Ser Glu Gly Asn Gly Ser Asp Pro 1 5 10 15 GAGCCG CCA GCC CCG GAG TCC AGG CCG CTC TTC GGC ATT GGC GTG GAG 576 Glu ProPro Ala Pro Glu Ser Arg Pro Leu Phe Gly Ile Gly Val Glu 20 25 30 AAC TTCATT ACG CTG GTA GTG TTT GGC CTG ATT TTC GCG ATG GGC GTG 624 Asn Phe IleThr Leu Val Val Phe Gly Leu Ile Phe Ala Met Gly Val 35 40 45 CTG GGC AACAGC CTG GTG ATC ACC GTG CTG GCG CGC AGC AAA CCA GGC 672 Leu Gly Asn SerLeu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly 50 55 60 AAG CCG CGC AGCACC ACC AAC CTG TTT ATC CTC AAT CTG AGC ATC GCA 720 Lys Pro Arg Ser ThrThr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala 65 70 75 GAC CTG GCC TAC CTGCTC TTC TGC ATC CCT TTT CAG GCC ACC GTG TAT 768 Asp Leu Ala Tyr Leu LeuPhe Cys Ile Pro Phe Gln Ala Thr Val Tyr 80 85 90 95 GCA CTG CCC ACC TGGGTG CTG GGC GCC TTC ATC TGC AAG TTT ATA CAC 816 Ala Leu Pro Thr Trp ValLeu Gly Ala Phe Ile Cys Lys Phe Ile His 100 105 110 TAC TTC TTC ACC GTGTCC ATG CTG GTG AGC ATC TTC ACC CTG GCC GCG 864 Tyr Phe Phe Thr Val SerMet Leu Val Ser Ile Phe Thr Leu Ala Ala 115 120 125 ATG TCT GTG GAT CGCTAC GTG GCC ATT GTG CAC TCG CGG CGC TCC TCC 912 Met Ser Val Asp Arg TyrVal Ala Ile Val His Ser Arg Arg Ser Ser 130 135 140 TCC CTC AGG GTG TCCCGC AAC GCA CTG CTG GGC GTG GGC TTC ATC TGG 960 Ser Leu Arg Val Ser ArgAsn Ala Leu Leu Gly Val Gly Phe Ile Trp 145 150 155 GCG CTG TCC ATC GCCATG GCC TCG CCG GTG GCC TAC CAC CAG CGT CTT 1008 Ala Leu Ser Ile Ala MetAla Ser Pro Val Ala Tyr His Gln Arg Leu 160 165 170 175 TTC CAT CGG GACAGC AAC CAG ACC TTC TGC TGG GAG CAG TGG CCC AAC 1056 Phe His Arg Asp SerAsn Gln Thr Phe Cys Trp Glu Gln Trp Pro Asn 180 185 190 AAG CTC CAC AAGAAG GCT TAC GTG GTG TGC ACT TTC GTC TTT GGG TAC 1104 Lys Leu His Lys LysAla Tyr Val Val Cys Thr Phe Val Phe Gly Tyr 195 200 205 CTT CTG CCC TTACTG CTC ATC TGC TTT TGC TAT GCC AAG GTC CTT AAT 1152 Leu Leu Pro Leu LeuLeu Ile Cys Phe Cys Tyr Ala Lys Val Leu Asn 210 215 220 CAT CTG CAT AAAAAG CTG AAA AAC ATG TCA AAA AAG TCT GAA GCA TCC 1200 His Leu His Lys LysLeu Lys Asn Met Ser Lys Lys Ser Glu Ala Ser 225 230 235 AAG AAA AAG ACTGCA CAG ACC GTC CTG GTG GTC GTT GTA GTA TTT GGC 1248 Lys Lys Lys Thr AlaGln Thr Val Leu Val Val Val Val Val Phe Gly 240 245 250 255 ATA TCC TGGCTG CCC CAT CAT GTC GTC CAC CTC TGG GCT GAG TTT GGA 1296 Ile Ser Trp LeuPro His His Val Val His Leu Trp Ala Glu Phe Gly 260 265 270 GCC TTC CCACTG ACG CCA GCT TCC TTC TTC TTC AGA ATC ACC GCC CAT 1344 Ala Phe Pro LeuThr Pro Ala Ser Phe Phe Phe Arg Ile Thr Ala His 275 280 285 TGC CTG GCATAC AGC AAC TCC TCA GTG AAC CCC ATC ATA TAT GCC TTT 1392 Cys Leu Ala TyrSer Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe 290 295 300 CTC TCA GAAAAC TTC CGG AAG GCG TAC AAG CAA GTG TTC AAG TGT CAT 1440 Leu Ser Glu AsnPhe Arg Lys Ala Tyr Lys Gln Val Phe Lys Cys His 305 310 315 GTT TGC GATGAA TCT CCA CGC AGT GAA ACT AAG GAA AAC AAG AGC CGG 1488 Val Cys Asp GluSer Pro Arg Ser Glu Thr Lys Glu Asn Lys Ser Arg 320 325 330 335 ATG GACACC CCG CCA TCC ACC AAC TGC ACC CAC GTG TGAAGGTTTG CGGGAG 1540 Met AspThr Pro Pro Ser Thr Asn Cys Thr His Val 340 345 CCTCCCGACT TCCAGCTCCCATGTGTGTTA GAGAGAGGAG GGCGGAGCGA ATTATCAAGT 1600 AACATGG 1607 ??

What is claimed is:
 1. A galanin receptor protein comprising an aminoacid sequence selected from the group consisting of an amino acidsequence represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 5 orits substantial equivalent thereto, or a salt thereof.
 2. The receptorprotein according to claim 1, which is produced by a transformant CHOcell.
 3. An isolated DNA which comprises a nucleotide sequence codingfor a galanin receptor protein of claim
 1. 4. A vector comprising theDNA according to claim
 3. 5. A transformant carrying the vectoraccording to claim
 4. 6. The transformant according to claim 5, whereinthe host cell is a CHO cell.
 7. A process for producing a galaninreceptor protein according to claim 1, which comprises culturing atransformant of claim 5 under conditions suitable to express saidgalanin receptor protein.
 8. A screening method for an agonist orantagonist of a galanin receptor protein according to claim 1, whichcomprises carrying out a comparison between: (i) at least one case wheregalanin is contacted with at least one component selected from the groupconsisting of a galanin receptor protein according to claim 1, a partialpeptide thereof and a mixture thereof, and (ii) at least one case wheregalanin together with a compound to be tested is contacted with at leastone component selected from the group consisting of a galanin receptorprotein according to claim 1, a partial peptide thereof and a mixturethereof.
 9. A kit for the screening of one or more agonists orantagonists to a galanin receptor protein according to claim 1, whichcomprises at least one component selected from the group consisting of agalanin receptor protein according to claim 1, a partial peptide thereofand a mixture thereof.
 10. An agonist or antagonist of a galaninreceptor, which is obtained by the screening method according to claim 8or the kit according to claim 9.